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Title Impaired T cell immunity in the Elderly via N-glycosylation

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Author Hayama, Ken Luke

Publication Date 2020

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Peer reviewed|Thesis/dissertation

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UNIVERSITY OF CALIFORNIA, IRVINE

Impaired T cell immunity in the Elderly via N-glycosylation

DISSERTATION

submitted in partial satisfaction of the requirements for the degree of

DOCTOR OF PHILOSOPHY

in Biomedical Sciences

Ken Luke Hayama

Dissertation Committee: Professor Michael Demetriou, Chair Professor Michael McClelland Professor Eric Pearlman

2020

DEDICATION

my parents,

Bakji Cho and Hatsuko Hayama,

and my brother Hiroki Hayama

TABLE OF CONTENTS

Page

LIST OF FIGURES iv

LIST OF TABLES v

ACKNOWLEDGMENTS vi

CURRICULUM VITAE viii

ABSTRACT OF THE DISSERTATION x

CHAPTER 1: Introduction 1

CHAPTER 2: Impaired T cell immunity in the elderly via interleukin-7 38 driven up-regulation of N-glycan branching

CHAPTER 3: Conclusions and Future Discussions 100

iii

LIST OF FIGURES

Page

Figure 1.1 IL-7 mediated signaling pathways 16

Figure 1.2 N-glycosylation branching pathway 17

Figure 1.3 Biosynthetic hexosamine pathway 18

Figure 2.1 N-glycan branching increases with age in mice 61

Figure 2.2 Lymphocytes from male do not exhibit increased N-glycan 63 Branching with age

Figure 2.3 Age dependent increase in N-glycan branching suppresses 65 T cell function and differentiation in mice

Figure 2.4 Regulating N-glycan branching in aged human PBMCs promote 67 T cell activity and proliferation

Figure 2.5 Genetically regulating N-glycan branching promotes immune 69 response to Salmonella in aged mice

Figure 2.6 Regulating N-glycan branching in aged male mice show minimal 71 effect to Salmonella infection

Figure 2.7 Regulating IL-7 signaling reduces N-glycan branching to 73 young phenotype

Figure 2.8 IL-7 signaling pathway increases with age to promote N-glycan 75 branching

Figure 2.9 N-glycan branching increases with age in humans 77

Figure 2.10 Age-dependent increase in N-glycan branching also suppresses 79 T cell activity in humans

Figure 2.11 IL-7 synergizes with age-dependent increase in endogenous 81 N-acetylglucosamine to increase N-glycan branching in humans

Figure 2.12 Inhibiting N-glycosylation in human cells reverse age-induced 83 suppression of T cell activity by N-glycan branching

LIST OF TABLES

Page

Table 2.1 Female and Male IL-7 Signaling Pathway Genes 85

Table 2.2 Female Only Young vs. Old Naïve DEGs 86

Table 2.3 Male Only Young vs. Old Naïve DEGs 88

Table 2.4 Female and Male Overlapping DEGs 91

Table 2.5 Female Glycosylation and related DEGs 92

ACKNOWLEDGMENTS

I would like to thank my thesis advisor, Dr. Michael Demetriou. He is a scholar who possesses profound scientific insight and a depth of experience in immunology who influenced and promoted my growth throughout my study. I’ll always remember his patience and kindness as I was graced with his mentorship. I am truly blessed to have been taken in as a member of his laboratory.

I would also like to thank the members of my advancement and dissertation committees, Dr. Michael McClelland, Dr. Eric Pearlman, Dr. Craig Walsh, and Dr.

Manuela Raffatellu. Their guidance and support throughout the years have been beneficial to my research. I am grateful for their support and dedication to provide feedback towards the completion of my study.

I would like to express my gratitude to the following members of the Demetriou lab for their friendship and support: Barbara Newton, Raymond Zhou, Lindsey Araujo,

Christie Mortales, Mike Sy, Sung-Uk Lee, Carey Li, and Khachik Khachikyan. I am also indebted to my undergraduate students, Kim Ly, Jasper Hai, Andrew Gong, Philip Lee, and Phuong Tran, who all performed research and experiments diligently. I would especially like to give special thanks to my lab mate Haik Mkhikian, whom has directly influenced and mentored me and guided this study from beginning to end. I would not have made much progress with my research without all the discussions and encouragement from my colleagues.

Lastly, I would like to give thanks to my parents and brother. Growing up my parents always encouraged me to pursue any topic that I am interested in. My brother

vi gave me the strength to strive towards graduate school by being the role model paving the way and showing anything is possible with hard work. Without their love and support

I would not have had the courage and tenacity to pursue higher education in the topics that I love. I am truly grateful for all the opportunities that they have provided me and I hope to instill the same values in my own family.

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CURRICULUM VITAE Ken Luke Hayama EDUCATION

UNIVERSITY OF CALIFORNIA, IRVINE ALIGN Irvine, CA Ph.D. in Biomedical Sciences 2020

UNIVERSITY OF CALIFORNIA, SAN DIEGO ALIGN San Diego, CA B.S. Biochemistry and Cell Biology 2009

HONORS AND AWARDS

NIH-BEST Professional Development Certificate ALIGN 2018 American Association of Immunologists Trainee Abstract Award 2017 American Association of Immunologists Trainee 2017 Bio-Techne Go Everywhere Travel Grant March 2017 Recipient 2017 UC Irvine School of Medicine Graduate Travel Award 2017 AAAS/Science Program for Excellence in Science 2015-2017 2nd place poster award – 13th UC Irvine Immunology Fair 2015 Francisco J. Ayala Graduate Teaching Fellowship 2014 Mentoring Excellence Certificate 2014 Howard Hughes Medical Institute Graduate Teaching Fellowship 2013 Howard Hughes Medical Institute Travel Award 2013

PEER-REVIEWED PUBLICATIONS

1. Hayama KL*, Mkhikian H*, Khachikyan K, Li CF, Zhou RW, Pawling J, Klaus S, Tran PQN, Ly KM, Gong AD, Saryan H, Hai JL, Lee PL, Rafatellu M, Dennis JW, and Demetriou M (2020). Impaired T cell immunity in the elderly via interleukin-7 and n-acetylglucosamine driven up-regulation of N-glycan branching. (To be submitted). * These authors contributed equally to this study 2. Brandt AU, Sy M, Lee SU, Newton B, Bellman-Strobl J, Pawling J, Li CF, Zimmermann H, Hayama KL, G Autreen, Rahman A, Yu Z, Kuhle J, Cooper G, Chien C, Scheel M, Dörr J, Wuerfel J, Dennis JW, Paul F, and Demetriou M (2020). N-Acetylglucosamine drives myelination and is associated with neuroprotection in multiple sclerosis. (To be submitted). 3. Mortales CL, Lee SU, Manousadjian A, Hayama KL, Demetriou M (2020). N-glycan branching decouples B cell innate and adaptive immunity to control inflammatory demyelination. iScience 23(8):101380. PMID: 32745987 4. Zocchi L, Wu SC, Wu J, Hayama KL and Benavente CA (2018). The cyclin- dependent kinase inhibitor flavopiridol (alvocidib) inhibits metastasis of human osteosarcoma cells. Oncotarget 9(34):23505-23518. PMID: 29805751

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5. Tajiri N, Quach DM, Kaneko Y, Wu S, Lee D, Lam T, Hayama KL, Hazel TG, Johe K, Wu MC and Borlongan CV (2017). NSI-189, a small molecule with neurogenic properties, exerts behavioral, and neurostructural benefits in stroke rats. J Cell Physiol 232(10):2731-2740. PMID: 28181668 6. White CA, Pone EJ, Lam T, Tat C, Hayama KL, Li G, Zan H and Casali P (2014). Histone deacetylase inhibitors upregulate B cell microRNAs that silence AID and Blimp-1 expression for epigenetic modulation of antibody and autoantibody responses. Journal of Immunology 193(12):5933-50. PMID: 25392531 7. Tajiri N, Quach DM, Kaneko Y, Wu S, Lee D, Lam T, Hayama KL, Hazel TG, Johe K, Wu MC and Borlongan CV (2014). Behavioral and histopathological assessment of adult ischemic rat brains after intracerebral transplantation of NSI-566RSC cell lines. PLoS One 9(3):e91408. PMID: 24614895 8. Li G, White CA, Lam T, Pone EJ, DC Tran, Hayama KL, Zan H, Xu Z and Casali P (2013). Combinatorial H3K9acS10ph histone modifications in IgH locus S regions target 14-3-3 adaptors and AID to specify antibody class switch DNA recombination. Cell Rep 5(3):702-714. PMID: 24209747

ABSTRACT OF THE DISSERTATION

Impaired T cell immunity in the Elderly via N-glycosylation

Ken Luke Hayama

Doctor of Philosophy in Biomedical Sciences

University of California, Irvine, 2020

Professor Michael Demetriou, Chair

Immunological function declines with age, increasing the susceptibility and severity of infections in the elderly. For example, those who are 65 years of age and older make up around 15% of the U.S. population but represents nearly 90% of the deaths associated with influenza. This age-associated reduction in immunity has been attributed to the dysfunction of both the innate and adaptive immune system. Indeed, functional capacity of T cells is severely diminished and is exacerbated with age. Our lab has revealed that

N-linked glycans play a critical role in controlling T cell immunity in mice and humans.

Furthermore, irregularity in N-glycosylation as a mechanism for dysfunction of T cells in the elderly has not been examined as of yet.

Here we report N-glycan branching in T cells increases with age in females, leading to T cell hypo-activity and increased susceptibility to infection. Reducing N-glycan branching rejuvenates T cell activation, proliferation and pro-inflammatory TH17 over anti- inflammatory Treg differentiation in aged female T cells. Susceptibility of aged female mice to Salmonella typhimurium invasion/dissemination was reduced by lowering N- glycan branching. A critical metabolic precursor of N-glycosylation and branching is N-

x acetylglucosamine (GlcNAc), a common amino sugar that is part of the regular human diet. GlcNAc is endogenous to human serum, increases with age and correlates with N- glycan branching in female > male human T cells. Interleukin-7 (IL-7) signaling synergistically regulates N-glycan branching with environmental GlcNAc. Both adoptive transfer of aged female T cells into young mice and negatively regulating IL-7 reversed aged-dependent increase in N-glycan branching. These results suggest that age- dependent increases of N-glycan branching, via increased supply of GlcNAc and IL-7 signaling, impairs T cell immunity in the elderly females, providing a novel mechanism to supplement current treatments for age-related diseases.

Chapter 1

Introduction

Portions of this chapter are taken from:

Mackall, C.L. et al. Harnessing the biology of IL-7 for therapeutic application. Nat Rev

Immunol 11, 330-342 (2011)

Reprinted with permission from Nature Publishing Group.

Aging is associated with a decline in immune function, leading to an increase in frequency and severity of infection in the elderly. This age-associated immune deficiency has been attributed to dysfunction in both innate and adaptive arms of the immune system. Existing peripheral T and B cells display reduced functional capacity with age.

Furthermore, there is a decrease in generating new naïve T and B cells in the elderly.

This issue is further exacerbated with the rapidly growing aging population. It is necessary that we develop a fundamental understanding underlying the cellular and molecular basis of immune aging in order to develop appropriate therapeutic measures to prevent age- related bacterial and viral infections such as the ongoing COVID-19 pandemic, and provide insight into the higher incidences of cancer related mortalities within the elderly population.

It has been recognized that the immune system is influenced by aging, but perhaps not always in the same direction1,2. Aging of the immune system is characterized by the paradox of two properties that incorporate different sides of the same coin – immunosenescence, which is commonly defined as the deterioration of immune function in relation to age, and inflammaging, which refers to a chronic inflammatory condition including elevated levels of pro-inflammatory chemokines and cytokines3. The mechanisms that underlie these age-related defects are not well understood and have been a focus of many recent studies due to advances in cellular and molecular phenotyping4. To further our understanding and inspire new ideas and directions towards immune aging this dissertation will introduce and explore the role N-glycosylation plays in T cell immunity of the elderly.

Immunosenescence

People worldwide are living longer, however the increase in lifespan does not coincide with a healthy life including being free from disability and chronic diseases. There is an unavoidable complex process involving a continuous compromise of a normally functioning immune system, suggesting that this immune dysfunction is represented not by a pre-established moment but rather a slow and steady process. This phenomenon called immunosenescence, a term coined by Roy Walford, affects both natural and acquired immunity5. Immunosenescence involves a restructuring of innate and adaptive immune functions. There is a decline in immune responses due to dysregulated immune responses, leading to severe consequences to bacterial and viral infections including reduced responses to vaccination2. Although innate immunity is considered relatively well preserved in the elderly, adaptive immunity is more susceptible due to age-associated functional decline and antigen burden that an individual is exposed to during their lifetime6. Various factors including genetics, nutrition, previous exposure to microorganisms, and steroid hormones differentially modulate the immune system7-9.

Consequently, infections and cancer occur more frequently within the elderly population.

While the overall number of circulating T cells remains relatively consistent with age, multifaceted changes occur that result in a decline of adaptive immune response with age. Critical changes in T cells that occur with advancement in age include decreased numbers of naïve T cells10-12, increased population of memory cells13,14, and decreased cellular proliferation and antigen response15,16. The developmental site of T cells, the thymus, atrophies with age, an phenomenon accelerating following onset of puberty17,18. This decline is due to defects in thymic stromal cell production of growth

3 factors and cytokines19,20. In an attempt to restore the size of the depleted population of peripheral T cells, existing populations of naïve T cells undergo homeostatic proliferation21,22. Thus, while the ratio between naïve and memory T cells begins high at a young age, it gradually reverses with age due to thymic involution combined with life- long immune responses to foreign pathogens. The long-term survival for these remaining

T cells are sustained by contact with cytokines and/or peptide-major histocompatibility complex (pMHC)23,24. Naïve T cells are sustained largely by interleukin-7 (IL-7), whereas memory T cell homeostasis requires both IL-7 and IL-1525.

Interleukin-7: mediator of homeostatic proliferation

IL-7 induces homeostatic proliferation of naïve T cells to maintain the naïve pool in adults following involution of the thymus. IL-7 is a member of the common gamma- chain (γc, CD132, IL2Rg) cytokine family, also identified as the IL-2 superfamily26, which comprises IL-2, IL-4, IL-7, IL-9, IL-15 and IL-2127 (Fig. 1.1). The receptors for these cytokines all share the same common gamma chain (γc, or CD132) while coming with cytokine-specific receptor subunits to form their unique heterodimeric receptor. An exception exists for IL-2R and IL-15R, which comprises a third shared β chain (CD122)28.

IL-7R comprises CD132 (γc) and its own unique α chain, CD127 (IL7Rα). The interleukins and ectodomains share very little sequence identity with each other, including their binding interfaces. Previous studies have shown that glycosylation does not play a role in complex formation between IL-2, IL-4, IL-6, IL-15, and IL-21 with their receptors29-33. IL-7 has three asparagines with six asparagines in the IL-7Rα ectodomain that may be N- glycosylated through their Asn-X-Ser/Thr (NXS/T, X≠P) recognition motif34. Although

4 previous studies have determined that glycosylation does not affect the binding or function of IL-7 to IL-7Rα35, it has been found that N-glycosylation outside the binding interface of IL-7/IL-7Rα indirectly accelerates the on-rate and binding36, suggesting other cytokine members may also be influenced indirectly through N-glycosylation. In addition to binding to IL-7, IL-7Rα also associates with thymic stromal lymphopoietin (TSLP) receptor to form a heterodimeric receptor complex that binds to TSLP37 . TSLP is an IL-

7-like cytokine that is also produced by stromal cells, in addition to epithelial cells and fibroblasts in the context of inflammation38. Numerous genomic studies have implicated

TSLP to allergic diseases including atopic dermatitis and increased risk of asthma39-42.

IL-7 is mainly secreted by stromal cells in lymphoid organs43-45. Current studies suggest that the production of IL-7 mRNA transcripts seem to occur at a fixed constitutive rate and is not influenced by extrinsic stimuli including feedback from IL-7 or the size of lymphocyte population46,47. Instead, the amount of available IL-7 is regulated by consumption by innate lymphoid cells (ILCs) and T cells rather than production48.

Typically, the amount of available IL-7 is sufficient to maintain a finite number of T cells.

After the lymphoid compartment is depleted, available T cells would encounter abundant

IL-7 and undergo homeostatic proliferation until equilibrium is re-established46. This increase in number of mature T cells result from increased proliferation of peripheral naïve

T cells rather than from enhanced thymic function49. The mechanism of production and availability of IL-7 seems to be unique and unlike other γc family members, such as IL-2 where production increases and decreases with T cell expansion and contraction respectively50.

Unlike IL-7 where there is a constant level of transcription, the expression of the gene encoding IL-7Rα is increased or decreased depending on the stage in T and B cell development. In addition, IL-7Rα expression in mature T cells are dramatically influenced by external stimuli including cytokines such as IL-751. IL-7 suppresses cell-surface expression of IL-7Rα in T cells by downregulating transcription of the gene encoding IL-

7Rα51. The amount of IL-7Rα on the cell surface plays a critical role in equilibrium by expressing higher levels in order to receive more signals by IL-7 for survival and proliferation. More IL-7Rα expressed results in more IL-7 consumed, therefore depriving neighboring cells of their signals. Since encountering IL-7 results in T cells transiently downregulating IL-7Rα expression, T cells would stop unnecessary consumption of IL-7, thereby regulating IL-7 availability for nearby cells to maintain a homeostatic population51,52. In addition to IL-7, other γc family members such as IL-2, IL-4, and IL-15 and even IL-6 which uses a receptor from a different family suppresses IL-7Rα expression51. TCR activation also resulted in inhibited IL-7Rα expression53-55.

IL-7 signaling pathways

IL-7 signals mainly through two different pathways: Jak-Stat (Janus kinase-Signal transducer and activator of transcription) and PI3K(phosphatidylinositol-3-kinase)/Akt. IL-

7 engaging IL-7Rα results in Jak3 selectively binding to the γc chain whereas Jak1 binds to IL-7Rα56-59. Mutations in either γc or Jak3 results in severe combined immunodeficiency (SCID) in humans60-64. The recruitment of Jak1 and Jak3 induces transphosphorylation which both increases their own intrinsic kinase activity and also the phosphorylation of the Y449 site on IL-7Rα65,66. The phosphorylated receptor then

6 becomes a docking site for Stat5 protein binding via SH2-phosphotyrosine interaction where Stat5 becomes phosphorylated by Jaks and dimerizes through their reciprocal phosphotyrosine-SH2 interaction67,68. Stat5 dimers are then exported into the nucleus to bind DNA69,70. The suppressor of cytokine signaling-1 (SOCS1) protein inhibits IL-7 signaling by either binding and inhibiting Jak1/3 through their kinase inhibitory region

(KIR), binding cytokine receptors to block Stat recruitment, or by functioning as ubiquitin ligases that mediate degradation of the signaling complex65,71-73. The absence of IL-7 signaling either by administering an anti-IL-7 neutralizing monoclonal antibody74, in IL-7-

/-75, IL-7Rα-/-76,77, γc-/-78, or Jak3-/-79 mice results in reduced thymic cellularity and lack of homeostatic proliferation.

The N-glycosylation pathway

All eukaryotic cells are surrounded by a dense layer of sugar chains and the proteins and lipids to which they are bound to, resulting in a structure collectively termed as the glycocalyx. The term originates from two Latin terms to describe its arrangement

– the first which is “glyco” literally translates to “sweet,” indicating the key building units consisting of various sugars and monosaccharides, such as mannose, glucose, and galactose. These sugars are connected in a plethora of combinations which vary in number, size, and complexity, forming the sugar conjugates known as glycans. Glycans are bound to proteins or lipids, resulting in a diverse array of glycoproteins, proteoglycans, and glycolipids. The term “calyx” translates into “outer coverings or husk.” This indicates the location as to where these sugars reside – extracellular to the cell membrane. With a dynamic and complex variety of cell-surface glycoproteins, the glycocalyx can vary in size

7 up to 100 nanometers thick, a size ten times wider than the plasma membrane. The glycocalyx dominates cell surface topology, suggesting that these glycans play a significant role in regulating cellular communication and function.

Two major classes of glycoproteins are defined accordingly to what the glycan is attached to on the protein. If the glycan is attached via nitrogen (N) in the asparagine side chain, then it is defined as N-glycans. On the other hand, if the glycan is attached via oxygen (O) located within a serine or threonine side chain, then it is defined as an O- glycan. A majority of cell surface receptors and transporters are co- and post- translationally modified with these N-linked and O-linked glycans in the endoplasmic reticulum (ER) with further modifications occurring within the Golgi apparatus80,81.

Modifications of these glycans are achieved by a series of sequential glycohydrolase and glycosyltransferase-mediated reactions resulting in multiple glycoforms that differ in binding avidity to sugar-binding proteins such as galectins (Fig. 1.2).

N-glycosylation of proteins begins with the transfer of a pre-assembled glycan,

Glc3Man9GlcNAc2 (three glucoses, nine mannoses, and two N-acetylglucosamines), by oligosaccharyltransferases within the endoplasmic reticulum to the asparagine (Asn) residue of Asn-X-Ser/Thr (NXS/T, X≠P) motifs82. The newly added glycan is then modified by the removal of three glucose molecules by glucosidase I (GI) and glucosidase II (GII).

Further modifications occur during transit through the Golgi via removal of mannose

(Man) residues from the trimmed glycan, Man9GlcNAc2, by Mannosidase I (MI). This results in the remaining Man5GlcNAc2 glycan structure which is the starting substrate for the first N-acetylglucosaminyltransferase (Mgat) enzyme (Fig. 1.2)83.

The Golgi N-acetylglucosaminyltransferase enzymes I, II, IV and V (encoded by

Mgat1, Mgat2, Mgat4a/b and Mgat5) act in a sequential order to initiate N-glycan branching by catalyzing the addition of N-acetylglucosamine (GlcNAc) from uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) onto mannose molecules to form N- glycan intermediates. The first N-glycan intermediate is formed through the addition of

GlcNAc by Mgat1, creating a mono-antennary N-glycan structure84. Mannosidase II (MII) modifies this initial structure by removing two mannoses to make room for Mgat2 to add an additional GlcNAc to form a bi-antennary N-glycan structure. Mgat4 and Mgat5 may further modify these N-glycans by catalyzing the addition of another GlcNAc molecule onto mannoses, resulting in a tri- and tetra-antennary N-glycan structure respectively.

Although these Golgi branching enzymes utilize the common sugar-nucleotide substrate

GlcNAc, they do so with declining efficiency such that both Mgat4 and Mgat5 activity are limited by the metabolic production of UDP-GlcNAc. Thus, activity of Golgi enzymes and the availability of substrates combine to determine the degree of N-glycan branching.

These GlcNAc branches can be extended with the addition of galactose (Gal) by galactosyltransferase-3 (GalT3) to form the disaccharide N-acetyllactosamine (LacNAc).

These LacNAc units are binding ligands for galectin family of sugar binding proteins and can be extended to synthesize poly-N-LacNAc via alternating actions of the addition of

GlcNAc by β1,3-N-acetylglucosaminyltransferase-2 (β3GnT2, an i-extension enzyme iGnT) and galactose by GalT385,86. LacNAc branches and/or extensions can be further modified or terminated through fucosylation and sialylation80,87-89. The plant lectin L-PHA

(Phaseolus vulgaris, leukoagglutinin) specifically binds to β1,6 branched N-glycans, a product of Mgat5, and can be utilized as a sensitive marker for determining levels of N-

9 glycan branching at the cell surface. Another plant lectin, LEA (Lycopersicon esculentum), recognizes and binds to poly-N-LacNAc structures and can be utilized to assess the degree of branching extension.

Regulation of N-glycan branching by the hexosamine biosynthetic pathway

The hexosamine biosynthetic pathway competes with glycolysis for available glucose to synthesize the sugar-nucleotide uridine diphosphate-N-acetylglucosamine

(UDP-GlcNAc), the substrate utilized by the Golgi branching enzymes (Fig. 1.3). This pathway merges with glycolysis by utilizing fructose-6-phosphate to form glucosamine-6- phosphate. Although glucose is mainly metabolized through glycolysis, about 2-5% enters the hexosamine pathway to synthesize UDP-GlcNAc de novo. Only a small percentage of fructose-6-phosphate is converted to glucosamine-6-phosphate due to the rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFAT). Alternatively, GlcNAc can also be directly salvaged into the hexosamine pathway via phosphorylation by N- acetyl glucosamine kinase (NAGK) to generate UDP-GlcNAc, thereby bypassing the rate- limiting first step of the hexosamine biosynthetic pathway.

In addition to differing Golgi enzyme activity responsible for N-glycan modifications, regulating biosynthesis of UDP-GlcNAc supply through the hexosamine pathway results in a versatile and dynamic glycoforms differing between the same proteins. Since N-glycan branching is dependent on the supply of UDP-GlcNAc via the hexosamine pathway, metabolic supplementation with GlcNAc should enhance N-glycan branching. Indeed, supplementing cells with GlcNAc with/without uridine has been shown to effectively increase surface N-glycan branching90. Furthermore, GlcNAc can be used

10 alone or with other immune-regulatory drugs as it has been used as a dietary supplement for years without reported adverse effects91. GlcNAc enters the cells through micropinocytosis, a process that is dependent upon the rate of membrane turnover.

Therefore, GlcNAc efficiency of enhancing surface N-glycan branching is increased in rapidly proliferating cells that display high amounts of membrane turnover in comparison to resting cells.

The galectin-glycoprotein lattice

Galectins are a family of carbohydrate binding proteins that are ubiquitously expressed at the cell surface. Each of these galectins have a carbohydrate recognition domain (CRD) which binds to LacNAc units on glycans92. In mammals, there are 15 known galectins that interact with multivalent glycan ligands on glycoproteins to form molecular lattices. These soluble galectins have been categorized into three structural groups defined as prototypical, tandem-repeat, and chimeric galectins93,94. The first prototypical group contains a single CRD and consists of galectin-1, -2, -5, -7, -10, -11, -

13, -14, and -15. Although they possess a single CRD, they frequently dimerize, similar to tandem-repeat galectins which contain two CRDs and consists of galectin-4, -6, -8, -9, and -1295. The chimeric galectin -3 contains a single CRD that can form a pentamer in the presence of multivalent ligands96.

The binding avidity between galectins and glycoproteins is proportional to the number of LacNAc units that is determined by the number of N-glycan sites, the degree of N-glycan branching (mono- to tetra-antennary) and the degree of branching extension of poly-N-LacNAc. The aforementioned is regulated by the expression and efficiency of

Golgi enzymes and their respective substrates. Thus, binding avidity between galectins and glycoproteins increase in proportion to LacNAc content produced by N-glycan Golgi enzymes, a factor established by genetic (enzyme expression and activity) and metabolic

(UDP-GlcNAc substrate quantity) factors. Galectins binding to N-glycan ligands via

LacNAc structures forms the galectin-glycoprotein lattice which regulates receptors and transporters via lateral movement, clustering, membrane localization and cell surface retention/endocytosis97.

Regulating T cell growth and signaling by N-glycan branching

The plasticity of T cell immune response is regulated by an intricate interplay of co-stimulatory and co-inhibitory signals to benefit the host while preventing any aberrant behaviors. T cell activation and proliferation is initiated through the clustering of a threshold number of T cell receptors (TCRs) at a specialized contact site, an immunological synapse, where the TCRs recognize foreign peptides bound to the major histocompatibility complex (MHC) site displayed on the surface of the antigen-presenting cell (APC)98-100. The number of TCRs required for clustering to initiate T cell activation is decreased by co-stimulatory signaling by CD28 to CD80/CD86 on APCs101,102. The galectin-glycoprotein lattice however negatively regulates T cell activation through the binding of multivalent galectins, such as galectin-1 and galectin-3, to N-glycans of the

TCR complex by limiting lateral movement and clustering in response to ligands. The binding affinity of TCR for peptide-MHC is similar to that of galectins to LacNAc units in

N-glycans, suggesting that the peptide-MHC complex must overcome galectin-TCR interactions in order to induce T cell response103. Indeed, by reducing N-glycan branching

12 through deletion of N-glycan Golgi enzymes Mgat1, Mgat2, Mgat5, or poly-LacNAc extending β3GnT2 reduces binding avidity of galectins to TCRs, resulting in robust T cell activation104-110 but also promotes dysregulation of T cell activation and autoimmune disorders110-112. Consistent with the aforementioned, removal of the encoded N-glycan sites within the TCR also results in an increase in T cell activation113. Collectively, N- glycan branching defines the limits of TCR affinity for peptide-MHC interactions essential for T cell activation.

Regulating the T cell response through inhibitory signaling is critical for T cell self- tolerance and growth arrest. Cytotoxic T-lymphocyte associated protein 4 (CTLA-4,

CD152) is an inhibitory receptor that is expressed at the cell surface 4-5 days after T cell activation to induce growth arrest114. CTLA-4 competes with CD28 for CD80/CD86 co- stimulatory ligands expressed on APCs to negatively influence T cell proliferation.

Transcriptional levels and intracellular stores of CTLA-4 increase in proportion to TCR signaling during early T cell activation. However, endosomal trafficking limits expression of CTLA-4 on the cell surface, resulting in it being predominantly found within the endosomes. On the other hand, CD28 is constitutively expressed on the cell surface of T cells, although it has a weaker avidity to CD80/CD86 in comparison to CTLA-4. In addition, CTLA-4 has a low number of N-glycan sites (two NXS/T motifs, where X≠P) and is therefore highly sensitive to metabolic supplement of UDP-GlcNAc and upregulation of

N-glycan branching. Indeed, T cells deficient in Mgat5 have decreased levels of CTLA-

4105. However, strong co-stimulation with CD28 and enhanced surface N-glycans through supplementation of UDP-GlcNAc resulted in similar surface levels of CTLA-4 in activated

Mgat5-/- and Mgat5+/+ T cells83,115,116. Galectins bound to upregulated N-glycans attached

13 to CTLA-4 enhanced surface retention by opposing endocytic loss, thereby promoting and sustaining growth arrest signaling. In addition to CTLA-4, programmed cell death protein 1 (PD-1) is an immune checkpoint inhibitor that fine-tunes T cell responses and is a major regulator of T cell exhaustion117. Our lab has shown that N-glycan branching markedly enhances PD-1 surface expression and blocking N-glycan branching with small molecule inhibitor kifunensine (which targets MI) in human CD4+ T cells reduces expression of PD-1 on the cell surface (unpublished data). Notably, the expression of PD-

1 increases on T cells of older individuals and inhibiting it partially restores T cell functional competence118-120. Blocking immune suppression via CTLA-4 and PD-1 are currently popular FDA approved treatments for cancer immunotherapy121-123.

Following growth arrest, activated T cells differentiate into effector T cells dependent on induction of certain transcription factors in response to TCR signal strength124,125 coupled with cytokines produced and secreted by an activated innate immune system. Under non-polarizing conditions (i.e. absence of exogenous cytokines),

+ N-glycan branching promotes differentiation of CD4 T cells into anti-inflammatory TH2

126,127 cells (secretes IL-4) while inhibiting pro-inflammatory TH1 cells (secrets IFNγ) .

Blocking N-glycan branching via Mgat1 deletion eliminates the ability of CD4+ T cells to differentiate into Tregs104. N-glycan branching drives induced Treg differentiation by promoting IL-2Rα (CD25) surface expression and signaling in blasting CD4+ T cells. In addition, N-glycan branching also critically suppresses pro-inflammatory TH17

104 differentiation . TH17 inducing cytokines (TGFβ+IL-6+IL-23) down-regulates branching in blasting CD4+ T cells, resulting in lowered IL-2Rα surface expression and associated

IL-2 signaling to promote TH17 differentiation over iTreg. Reversing lowered branching

14 caused by TH17 induction by supplementing GlcNAc blocks TH17 differentiation and induces a cell fate switch to iTregs by restoring IL-2Rα and thus IL-2 signaling.

T cell dysfunction is influenced by many factors including disruption of glycosylation at the cell surface. Our lab has shown that in T cells, N-glycan branching globally influences T cell activation and differentiation. Given the importance of N-glycan branching in negatively regulating T cell function it is a surprise that nothing is known of their role in immune senescence. Here we explore for the first time how N-glycan branching is involved in T cell immune responses in the elderly.

Figure 1.1 IL-7 mediated signaling pathways. IL-7 signals through the IL-7 receptor, a heterodimer consisting of IL-7Rα and the common γ chain. During T cell development within the thymus, IL-7 signaling participates in TCR gene rearrangement by histone acetylation and DNA demethylation. After maturation, IL-7 mediated signaling initiates downstream signaling pathways by interacting with JAK1, JAK3, and PI3K, resulting in the phosphorylation and activation of STAT5. This results in up and downregulated expression of multiple genes responsible for survival and proliferation. IL-7 signaling can also leads to reduced expression of p27 and increased expression of CDC25A, resulting in further upregulation of proliferation. Some of the effects of IL-7 can also be substituted for by having IL-7Rα complexed with thymic stromal lymphopoietin (TSLP) receptor (TSLPR). Abbreviations: MHC, Major Histocompatibility Complex; IL-7, Interleukin-7; IL- 7α, Interleukin-7α (also known as CD127); γc, common cytokine receptor γ–chain (also known as CD132); TCR, T cell receptor; Ac, histone acetylation; me, DNA demethylation; γδ TCR, gamma delta T cell receptor; αβ TCR, alpha beta T cell receptor; CBL-C, Casitas B-lineage lymphoma B; PI3K, phosphoinositide 3-kinase (PI3K); JAK1, Janus kinase 1; JAK3, Janus kinase 3; p27, cyclin-dependent kinase inhibitor 1b (also known as p27Kip1); CDC25A, cell division cycle 25 homologue A; AKT, protein kinase B; STAT5, signal transducer and activator of transcription 5; BCL-2, B cell lymphoma 2; MCL1, myeloid cell leukemia sequence 1; BAX, BCL-2-associated X protein; BIM, BCL-2-interacting mediator of cell death; BAD, BCL-2 antagonist of cell death. Image © Mackall C.L. et al. Nature Reviews Immunology Volume 11, pages 330-342 (2011)

Figure 1.2 N-glycosylation branching pathway. Oligosaccharyltransferase transfers a pre-assembled glycan, Glc3Man9GlcNAc2, to the NxS/T motifs (Asp-X-Ser-Thr where X ≠ Pro) on glycoproteins within the endoplasmic reticulum (ER). These glycans are further modified by mannosidases (MI and MII) and N-acetylglucosaminyltransferase (Mgat) enzymes, which act sequentially to generated N-glycans that display increasing avidities for galectins. Mgat1, 2, 4, and 5 utilizes the same sugar-nucleotide donor, UDP-GlcNAc, but with declining efficiency such that both Mgat4 and Mgat5 are limited for branching by the metabolic production of UDP-GlcNAc. Galactosyltransferase 3 (GalT3) and iGnT further extends the branches by adding galactose to GlcNAc, creating N- acetyllactosamine (LacNAc) which can be bound by galectins. Small molecular inhibitors kifunensine (KIF) and swainsonine (SW) can be used to eliminate or reduce branching respectively. Plant lectins L-PHA (Phaseolus vulgaris, leukoagglutinin) and LEA (Lycopersicon Esculentum lectin) binding sites are also shown. Abbreviations: OT, oligosaccharyltransferase; GI, glucosidase I; GII, glucosidase II; MI, mannosidase I; MII, mannosidase II; Mgat, N-acetylglucosaminyltransferase; GalT3, Galactosyltransferase 3; iGnT, i-branching enzyme b-1,3-N-acetylglucosaminyltransferase; KIF, kifunensine; SW, swainsonine; GlcNAc, N-acetyl glucosamine; UDP, uridine diphosphate; UMP, uridine monophosphate; Km, Michaelis constant of the enzyme.

Figure 1.3 Hexosamine Biosynthetic Pathway. Glucose enters the cell de novo through a glucose transporter and is metabolized. After being converted into fructose-6- phosphate, the substrate has an option of continuing through glycolysis. However, the hexosamine biosynthetic pathway merges from here to form glucosamine-6-phosphate by the rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFAT) by utilizing glutamine as an amino donor128. To produce N-acetylglucosamine-6-phosphate, Glucosamine-6-phosphate can either be acetylated or GlcNAc can be salvaged via micropinocytosis and enter the hexosamine pathway by N-acetylglucosamine kinase (NAGK). N-acetylglucosamine-6-phosphate is further isomerized to N- acetylglucosamine-1-phosphate and is ultimately activated into UDP-N- acetylglucosamine (UDP-GlcNAc), the common precursor for glycosylation. Abbreviations: PFK, phosphofructokinase; GFAT, glutamine fructose-6-phosphate amidotransferase; CoA, coenzyme A; GlcNAc, N-acetylglucosamine; NAGK, N- acetylglucosamine kinase; UTP, uridine triphosphate; PPi, inorganic pyrophosphate; UDP, uridine diphosphate.

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Chapter 2

Impaired T cell immunity in the elderly via interleukin-7 driven up-regulation of

N-glycan branching

Summary

Impaired T cell immunity in the elderly increase’s mortality from infectious disease.

The branching of Asparagine (N) - linked glycans serves as a critical negative regulator of T cell immunity. Here we report that branching in female > male and naïve > memory

T cells increases with age. Reversing this increase rejuvenates T cell function and limits severity of Salmonella infection. Naïve T cells are generated by the thymus in adult mice, but by interleukin-7 (IL-7) driven homeostatic proliferation in adult humans. Co-incident with thymic involution in old mice, IL-7 signaling increased in female naïve T cells, which in turn increased branching. Serum levels of N-acetylglucosamine, a rate limiting metabolite for branching, increased with age in humans and synergized with IL-7 to raise branching. Thus, in the elderly naïve T cell generation via IL-7 combines with rising N- acetylglucosamine to secondarily suppress function via branching. Targeting this pathway may reduce severity of infections like COVID-19 in the elderly.

Introduction

Aging is associated with diminishing immune functional response, contributing to the increased morbidity and mortality rate from neoplastic diseases and infections. Within the United States approximately 90% of influenza associated deaths occur among adults over the age of 65, despite this age group representing only 15% of the population1-4. The vulnerability of the elderly to infectious agents have been recently highlighted with the emergence of SARS-CoV-2, the virus responsible for the COVID-19 pandemic5. This increase in morbidity and mortality among elderly patients extends toward common bacterial infections including Salmonella gastroenteritis6. With the elderly population rapidly increasing in numbers, interventions that effectively rejuvenate the immune response is essential to prevent or regulate age-related pathologies to maintain or improve the health status of society.

Age-associated decline in immune function, or immune senescence, has been attributed to the dysfunction of both the innate and adaptive arms of the immune system.

The generation of naïve T and B lymphocytes decline with age, whereas the functional capacity of existing peripheral T and B cells are largely reduced among the elderly7,8.

Previous transcriptomic profiling of aging in naïve and memory CD4+ T cells from mice through microarray data analysis provide potential molecular targets which may drive age-related functional decline in aged immune cells9-11. In addition, global gene expression profiling in humans show conserved alternations between mouse and human data sets where dysregulation of basal NF-κB may contribute to IL-7 responsiveness in aging12-14. Despite the identification of multiple contributing deficiencies in immune

38 senescence, the molecular underpinnings of age-dependent impairment of T cell functions are not well understood.

Asn (N) – linked glycans plays a critical role in regulating T cell immunity in mice and humans15-19. Virtually all cell surface and secreted proteins are co- and post- translationally modified by the addition of complex carbohydrates in the ER/Golgi secretory pathway. These glycans impart substantial molecular information that is not encoded by the genome, thus providing an avenue for versatility by not being restricted to abide by genetic templates. Previous studies done in our lab have revealed that sugar binding proteins, such as galectins, bind to glycans on TCRs and other receptors and transporters at the cell surface, forming a molecular lattice that controls clustering, signaling and endocytosis of these proteins to affect cell growth, differentiation and thus immune response in mice and humans15-27. In T cells, N-glycan branching cooperates with the galectin lattice to negatively regulate TCR clustering and signaling15,16, promotes surface retention of CTLA-4, PD-1 and transforming growth factor-β receptor (TβR) type

16,26,28 I and II , inhibits TH1 and TH17 while promoting TH2 and induced T regulatory (iTreg) cell differentiation20,21,24 and suppresses development of autoimmunity15,22,23 in mice and humans. N-glycan branching is metabolically regulated by Golgi branching enzymes utilizing a common sugar-nucleotide substrate, UDP-GlcNAc. However, they do so with declining efficiency such that metabolic production of UDP-GlcNAc is limited for branching by Mgat4 and 516. Thus, metabolic changes regulating biosynthesis of UDP-GlcNAc have profound effects on N-glycan branching.

Given the importance of N-glycan branching in negatively regulating T cell function and that immune function declines with age, we investigated whether N-glycan production

39 increases with age. Indeed, we demonstrate here that aging in female humans and mice is associated with increases in N-glycan branching in T cells and that reversing this phenotype rejuvenates T cell activity. Furthermore, this age-associated increase is driven by the IL-7 pathway, thus providing a potential therapeutic target for immune senescence.

Materials and Methods

Mice and human subjects

C57BL/6J (000664), Mgat2f/f (006892), Lck-Cre (003802), and Cd45.1 Pep Boy (002014) mice were obtained from Jackson Laboratory. Young and aged C57BL/6 mice were obtained from the National Institute of Aging (NIA) maintained at Charles River Laboratories (Wilmington, MA, USA). Upon arrival mice were allowed to acclimate to the new environment for at least one week prior to the start of an experiment. To acquire mouse plasma, blood was collected in sterile tubes containing 0.1M EDTA as an anticoagulant. All human subjects are Caucasian (non-Hispanic) and include both male and females. Individuals with cancer, uncontrolled medical disease or any other inflammatory syndrome were excluded. Human whole blood was collected from healthy individuals through the Research Blood Donor Program serviced by the Institute for Clinical & Translational Science (ICTS) or through The 90+ Study at the University of California, Irvine (UCI). All procedures with human subjects were approved by the Institutional Review Board of the University of California, Irvine.

T cell isolation and culture

For all in vitro mouse experiments, erythrocytes were depleted from splenic or lymph node cell suspensions by incubation with red blood cell (RBC) lysis buffer followed by negative selection using EasySep™ Naïve CD4+ T Cell Isolation Kit or EasySep™ CD4+ T Cell Isolation Kit (STEMCELL Technologies) according to manufacturer’s instructions and supplemented with or without 20μg/mL biotinylated Phaseolus Vulgaris Leucoagglutinin (L-PHA, Vector Labs) to deplete non-Mgat2 deleted cells. Freshly harvested cells were cultured (2x105-5x106 cells) in RPMI-1640 (Thermo Fisher Scientific) supplemented with 10% heat inactivated fetal bovine serum (Sigma Aldrich), 2μM L-glutamine (Gibco), 100U/mL penicillin/streptomycin (Gibco), and 50μM β-mercaptoethanol (Gibco) as previously described 15,16,19.

Human peripheral blood mononuclear cells (PBMCs) isolated by density gradient centrifugation over Histopaque-1077 (Sigma-Aldrich) or Lymphoprep (StemCell Technologies) were stimulated with plate-bound anti-CD3 (OKT3, eBioscience) plus soluable anti-CD28 (CD28.2, eBioscience) in media as described above. PBMCs were pre-treated with or without 5μM KIF 2 days prior to stimulation if indicated. For pharmacological treatments, cells were incubated with GlcNAc (40mM added daily, Wellesley Therapeutics), kifunensine (5uM, Glycosyn), recombinant human IL-7 (0.5ug- 1.5ug, R&D Systems), and/or anti-mouse/human IL-7 antibody (clone M25; 5-15ug,

BioXCell) as indicated. TH17 and iTreg induction were performed as previously described20.

Flow Cytometry

Flow cytometry experiments were performed as previously described 15,19. Mouse antibodies for surface, intracellular and phospho-flow staining to CD69 (H1.2F3), IL17 (eBio17B7), CD4 (RM4-5), CD8α (53-6.7), CD19 (1D3), Foxp3 (Fjk-16s), CD44 (IM7), CD62L (MEL-14), CD3 (145-2C11 and 17A2), IL7Rα (A7R34), Rat IgG2a kappa Isotype Control (eBR2a), Phospho-STAT5 (SRBCZX), Mouse IgG1 kappa Isotype Control (P3.6.2.8.1) were purchased from eBioscience. In addition, CD45RA (HI100), CD4 (RM4- 5) and CCR7 (4B12) were purchased for human cells. DyLight 649 conjugated Streptavidin (SA-5649) and both Fluorescein labeled and biotinylated L-PHA were purchased from Vector Labs. Anti-mouse CD45.1 (A20) and CD45.2 (104), and anti- human CD45RO (UCHL1) and CD8 (SK1) were purchased from BioLegend. Proliferation was assessed by staining cells with CellTrace CFSE proliferation kit (Thermo Fisher Scientific). Flow cytometry experiments were performed with the BD FACSAria Fusion Sorter, LSR II, Invitrogen Attune NxT Flow Cytometer, or ACEA Novocyte Flow Cytometer and analyzed following appropriate compensation using FlowJo software.

Mouse experiments

Mice were pre-treated with streptomycin (0.1 ml of a 200 mg/ml solution in sterile water) intragastrically prior to mock-infection or inoculation with a mixture of salmonella (Salmonella typhimurium, IR715 strain ATCC14028, 5×108 CFU/animal). At 72 hours after infection, mice were euthanized and the cecum, Peyer’s patches, mesenteric lymph nodes, and spleen were collected for analysis of immune cells. Analysis of bacterial dissemination were performed through serial 10-fold dilutions plated for enumerating bacterial CFUs on agar plates. Lymphocytes were prepared from pooled inguinal lymph nodes and splenocytes and purified for naïve CD4+ T cells from CD45.2 C56BL/6 donor mice as described above then injected intravenously by tail vein in 200μl of phosphate-buffered saline (PBS) into CD45.1 congenic C57BL/6 recipient mice. For in vivo IL-7 treatment, mice were i.p. injected with either 200µl of PBS or rhIL-7/M25 cytokine/antibody complex (R&D Systems and BioXCell) every two days. Before injection, the cytokine/antibody complex were generated through co-incubation for 30 mins at room temperature. GlcNAc was administered orally by adding GlcNAc to their drinking water at 1mg/ml. Fresh GlcNAc was given daily for the duration of the experiment. For IL-7 blockade experiments, mice were i.p. injected with 1-1.5mg of mouse IgG1 isotype control antibody (MOPC-21, BioXCell) or anti-mouse/human IL-7 (M25, BioXCell) in 200µl of PBS three times at 2 day intervals per week as indicated.

RNA extraction, library prep, and sequencing

Lymphocytes were prepared from pooled lymph nodes (axillary, brachial, cervical, inguinal) and splenocytes then purified for CD4+ T cells through negative selection by EasySep™ CD4+ T cell Isolation Kit (STEMCELL Technologies). Cell suspensions are then stained and FACS-sorted for either naïve (CD3+CD4+CD25-CD62L+CD44-) or memory (CD3+CD4+CD25-CD62L-CD44+) populations by BD FACSAria Fusion Sorter. Total RNA were then isolated using RNeasy Plus Micro Kit (Qiagen) and analyzed for RNA integrity number (RIN) by Agilent Bioanalyzer 2100. RIN of all samples was

43 determined to be ≥9.0. Subsequently, 125ng of total RNA was used for library prep through NEBNext Poly(A) mRNA Magnetic Isolation Module before using NEBNext Ultra II DNA Library Prep Kit for Illumina. Collected samples was cleaned with AMPure XP beads (Beckman Coulter) to make RNA into strand specific cDNA libraries with multiplexing barcodes from NEBNext Multiplex Oligos for Illumina (Index Primers Set 1 and 2). RNASeq libraries were then analyzed by qPCR (KAPA Biosystem), normalized to 2nM and pooled for multiplexing in equal volumes, then underwent paired-end 100-bp sequencing run on HiSeq 4000 (Illumina). Statistical analysis for differential gene expression was performed using DESeq2.

LC-MS/MS analysis

Human serum samples for metabolomics analysis were prepared as described previously29. Metabolites from 50µl of serum were extracted by the addition of 200µl of ice-cold extraction solvent (40% acetonitrile, 40% methanol, and 20% water). Thereafter the samples were shaken at 4°C for 1 hour at 1400rpm in a Thermomixer R (Eppendorf) and then centrifuged at 4°C for 10 minutes at ~18,000 x g in an Eppendorf microcentrifuge. The supernatant were transferred to fresh tubes and evaporated in a Speedvac (Acid-Resistant CentriVap Vacuum Concentrators, Labconco). The dry-extract samples were stored at -80°C. The dry eluted metabolites were resuspended in 100ul of water containing internal standards D7-Glucose at 0.2mg/mL and H-Tyrosine at 0.02mg/ml. Eluted metabolites were analyzed in negative mode at the optimal polarity in MRM mode on electrospray ionization (ESI) triple-quadrupole mass spectrometer (AB Sciex 4000Qtrap, Toronto, ON, Canada). Standard curves were prepared by adding increasing concentrations of GlcNAc 13 or N-Acetyl-D-[UL- C6]glucosamine (Omicron Biochemicals) to 50µl aliquot of control serum. Raw data were imported to MultiQuant software (AB Sciex, Version 2.1) for peak analysis and manual peak confirmation. The resulting data including area ratio is then exported to Excel and analyzed with MetaboAnalyst 3.0.

Statistical analysis

All experimental data from in vitro and in vivo studies were analyzed for significance from at least three independent experiments. Correlation of age, sex, and serum GlcNAc were analyzed with a linear regression model for age. Correlations of serum GlcNAc levels with L-PHA staining on different cell populations were also analyzed with linear regression models. Statistical analyses were calculated with Prism software (GraphPad). P values were from two- or one-tailed unpaired t-tests unless specified otherwise.

Results

N-glycan branching increases with age in female mouse T cells

To explore whether N-glycan branching increases with age, we initially compared

L-PHA binding (Phaseolus vulgaris, leukoagglutinin) in splenic mouse T cells from young

(~4-5 months) and old (~20-23 months) adult mice. L-PHA binds to β1,6GlcNAc-branched

N-glycans and is utilized as a sensitive quantitative marker for N-glycan branching15.

Indeed, flow cytometry revealed an increase in female CD4+ T cells, with differences in naïve > central memory > effector memory populations (Fig. 2.1A-C, 2.2A). Remarkably, naïve CD4+ T cells displayed nearly a 2-fold increase in L-PHA binding, whereas only a

~20% difference is sufficient to alter T cell function and risk for developing inflammatory diseases22,23. In comparison, L-PHA binding in old male CD4+ T cells was not elevated

(Fig. 2.1D) and was lower than old female CD4+ T cells, indicating that branching specifically increased in elderly female but not male mice relative to young mice (Fig.

2.2B,C). This extended to male CD8+ T cells displaying no difference, whereas female

CD8+ naïve T cells demonstrated a modest increase in L-PHA binding but not in the memory subsets (Fig. 2.1E,F).

CD19+ B cells in both young and old females and males displayed little difference in N-glycan branching (Fig. 2.2D). Age-associated thymic involution involves a decrease in cellularity, ultimately resulting in a reduction in naïve T cell output30-32. Although N- glycan branching regulates T cell production and development from the thymus19, single and double positive thymocytes demonstrated no age-associated differences in L-PHA binding (Fig. 2.2E). This indicates that the increase in N-glycan branching in old naïve female T cells occurs in the periphery rather than from alterations in thymocyte

46 development. Together, these data indicate an increase in N-glycan branching with age in female but not male mouse T cells, with the most striking differences being displayed within the naïve CD4+ T cell subsets.

Age-associated increase in N-glycan branching suppresses T cell function

N-glycan branching negatively regulates TCR clustering and signaling, thus limiting T cell immune response. As N-glycan branching increases with age, we explored

T cell activation thresholds by comparing induction of the T cell activation marker CD69 in response to anti-CD3 antibody. Indeed, CD4+ T cells from aged female mice were hypo- reactive in comparison to young CD4+ T cells (Fig. 2.3A). This effect was reversed by supplementing the cells with mannosidase I inhibitor kifunensine (KIF), which blocks N- glycan branching18, thus confirming that the elevated levels in N-glycan branching in old

T cells inhibited ligand induced TCR signaling (Fig. 2.3A, Fig. 2.4A). We further confirmed this phenotype by examining aged mice with T cell specific deletion of the

Mgat2 Golgi branching enzyme (i.e. Mgat2fl/fllck-cre mice). T cells lacking Mgat2 results in N-glycans with a single branch that is extended by poly-N-acetyllactosamine17. Indeed, old Mgat2 deficient CD4+ T cells display up-regulation of CD69 relative to age-matched control CD4+ T cells (Fig. 2.3B). Consistent with these results, proliferation of old female

CD4+ T cells was also rejuvenated by decreasing branching with KIF or by genetic deletion of Mgat2 (Fig. 2.3C).

In addition to limiting TCR activity, N-glycan branching suppresses inflammatory T cell responses by inhibiting pro-inflammatory TH17 differentiation while promoting anti- inflammatory Treg differentiation20,21. Therefore, the age-associated increase in N-glycan

47 branching is expected to reduce inflammatory responses by T cells. Indeed, reversing age-associated increases in N-glycan branching in old female CD4+ T cells via KIF or

Mgat2 deletion enhanced TH17 differentiation while reducing Treg induction (Fig. 2.3D,E).

Together, these results indicate that age-induced increase in N-glycan branching functionally suppresses T cell activity in females.

Reversing age-induced increases in N-glycan branching rejuvenates immunity to

Salmonella typhimurium

In immune-competent hosts, non-typhoidal salmonella induces a robust TH17 response and a localized self-limited infection within the bowel mucosa33,34. However, salmonella infection in the elderly results in reduced TH17 induction and dissemination outside of the bowel mucosa6,35. To assess whether the age-associated increase in N- glycan branching suppresses T cell function in vivo, we explored an infection model with non-typhoidal salmonella (Salmonella typhimurium). We infected old wild type female mice with a virulent strain of salmonella (IR715), resulting in two deaths and significant dissemination from the bowel mucosa to the Peyer’s patches, mesenteric lymph nodes

(MLN), and the spleen (Fig. 2.5A-C). In contrast, infecting age-matched T cell specific

Mgat2 deficient female mice in parallel resulted in no deaths and significantly reduced dissemination to Peyer’s patches, MLN, and the spleen (Fig. 2.5A-C). Luminal content of

IR715 did not differ between wild type and Mgat2 deficient old female mice, suggesting a defect in dissemination of IR715 (Fig 2.6A). Consistent with previous data indicating a lack of increase in N-glycan branching in old male T cells, deletion of Mgat2 did not significantly alter IR715 dissemination and luminal content in old male mice (Fig. 2.5D-F,

Fig. 2.6B). In addition, our in vitro data indicates age-dependent increase in N-glycan branching suppresses TH17 while enhancing Treg responses where infected Mgat2 deficient old female mice displayed increased IL-17+ cells and reduced FoxP3+ cells with the bowel compared to infected wild type control mice. Collectively, age-dependent increase in N-glycan branching promotes Salmonella typhimurium dissemination by limiting TH17 response and promoting Treg responses in vivo.

IL-7 signaling pathway increases with age to raise N-glycan branching

Age-dependent increase in N-glycan branching in old naïve female T cells may arise from cell-intrinsic and/or cell-extrinsic factors. To assess this, we normalized the environment of naïve CD4+ T cells from young and old female mice to eliminate external factors. Indeed, culturing cells in the same media for three days was sufficient to markedly reduce the difference in N-glycan branching between young and old female naïve CD4+

T cells (Fig. 2.7A). Similarly, adoptively transferring old naïve CD4+ T cells that express

CD45.2 into young recipient CD45.1 female mice for two weeks resulted in significantly reduced differences in N-glycan branching between young and old naïve CD4+ T cells

(Fig. 2.7B, 2.8A). Together, these data suggest that cell extrinsic factors in the environment of aged female mice primarily drive age-associated increases in N-glycan branching.

To determine whether environmental driven changes in N-glycan branching is associated with changes in gene expression profiles of old female mice, we performed high throughput RNA sequencing (RNA-Seq) analysis on cell sorted young and old naïve

CD4+ T cells from female and male mice (Fig. 2.8B). Of the 158 and 192 differentially

49 expressed genes (DEGs) between young and old naïve CD4+ T cells in females and males, respectively, only 44 overlapped (Fig. 2.8C, Table 2.1-2.4). In addition, there were no differences observed in gene expression of N-glycan branching enzymes or other relevant genes involved in glycosylation (Table 2.5), which we confirmed by performing quantitative PCR for critical Golgi enzymes involved in N-glycan branching (Fig. 2.8D).

However, among the highest DEGs in females but not males included three within the IL-

7 signaling pathway (Table 2.1), namely reduced expression of IL7Rα (CD127) and increased expression of Suppressor of cytokine signaling 3 (Socs3) and Janus kinase 3

(Jak3). In contrast, other IL-7 signaling pathway genes were unchanged (Table 2.1). To confirm this RNA-seq data, we examined IL7Rα surface expression on naïve CD4+ T cells in females. Indeed, IL7Rα (CD127) protein is reduced in old female naïve CD4+ T cells relative to young female mice (Fig. 2.7C). Reduced IL7Rα and increased inhibitory Socs3 in old female mice is likely secondary to excessive IL-7 signaling36,37. Indeed, old naïve but not memory female T cells displayed increased basal expression of phosphorylated-

STAT5 relative to young controls (pSTAT5, Fig. 2.7D).

To examine whether increased levels of IL-7 signaling may enhance N-glycan branching, we cultured young and old naïve CD4+ T cells with recombinant IL-7. Indeed, supplementing IL-7 in vitro significantly increased N-glycan branching in both young and old naïve CD4+ T cells (Fig. 2.7E,F). To confirm whether the same effects can be achieved in vivo, we administered IL-7 complexed with the anti-IL-7 monoclonal antibody

M25 clone into young female mice. The IL-7/M25 complex significantly augments cytokine potency in vivo and improves IL-7 half-life38,39. Indeed, enhancing IL-7 signaling in vivo

50 by administering the IL-7/M25 complex at two different doses significantly raised N-glycan branching levels in naïve CD4+ T cells in young female mice (Fig. 2.7G).

IL-7 has a short serum half-life in mice (~2 hrs)38 and is not detectable by ELISA in the serum of old female mice (data not shown). Therefore, to investigate whether excessive IL-7 signaling in vivo raises N-glycan branching in naïve T cells of old female mice, we injected antagonistic doses of anti-IL-7 antibody (M25) to block endogenous IL-

7 signaling. Indeed, two weeks of IL-7 antagonism with M25 treatment reduced N-glycan branching levels in old naïve female CD4+ T cells to levels seen in young cells in both blood and the spleen (Fig. 2.7H,I). Furthermore, IL7Rα protein expression was partially restored following treatment in old naïve CD4+ T cells (Fig. 2.8E). As two weeks treatment to inhibit IL-7 signaling may have been too short to fully reverse the down-regulated expression of IL7Rα, we repeated the treatment for four weeks. Four weeks of M25 treatment reduced N-glycan branching similar to two weeks of treatment (Fig. 2.8F,G) as well as reversed the reduction in IL7Rα protein expression and elevated pSTAT5 levels previously observed (Fig. 2.7J,K). This confirms that excessive IL-7 signaling in vivo is responsible for both reduced IL7Rα and elevated basal pSTAT5 expression in old female mice. Together, our data indicates that increased basal IL-7 signaling in old female mice to induce homeostatic maintenance of the naïve CD4+ T cell population as a consequence of thymic involution due to age, secondarily raises N-glycan branching in naïve CD4+ T cells to diminish T cell function in old female mice.

IL-7 synergizes with N-acetylglucosamine to raise N-glycan branching in old human naïve T cells

In contrast to mice, where thymic involution occurs very late in life, in humans thymic production of naïve T cells begin decreasing from birth and significantly declines by puberty before ceasing at young adulthood31,40-42. Therefore, humans become highly dependent on IL-7 mediated homeostatic proliferation to maintain a naïve CD4+ T cell pool at much younger age compared with mice. Given this difference, we investigated whether N-glycan branching also increases with age in human T cells by examining females and males ranging in age from 19 to 98 years old. Surprisingly, we observed similar phenotypes as in mice, with age-dependent increases in N-glycan branching in

CD4+ T cells > CD8+ T cells and CD19+ B cells, with significantly bigger differences seen in females than male and naïve over memory subset populations (Fig. 2.9A-F, 2.10A-D).

Restricting analysis to ages between 19 and 65 showed similar results in female CD4+ T cells, indicating that the data is not skewed by those above 90 years of age and instead indicates a gradual and consistent increase in N-glycan branching with age (Fig.

2.10E,F). As seen in mice, age-associated increases in N-glycan branching suppressed

T cell activity, as treating old female PBMCs with KIF to inhibit N-glycan branching rejuvenated ligand-induced T cell activation and proliferation (Fig. 2.12A,B).

Our lab has previously reported that short-term treatment of CD4+ T cells (mixed naïve and memory populations) for 3 days with recombinant IL-7 lowers branching, which is opposite of what we have observed with mouse naïve CD4+ T cells. However, this analysis did not consider gender differences or naïve versus memory T cells. In addition, homeostatic maintenance of the naïve T cell pool involves prolonged exposure to IL-7 in vivo, therefore we repeated these experiments for 6 days and analyzed naïve female

CD4+ and CD8+ T cells at rest. This revealed that exogenous IL-7 induced little, if any,

52 increase in N-glycan branching in naïve CD4+ T cells and a small increase in naïve CD8+

T cells.

Our lab has previously shown that IL-7 regulates mRNA expression of the Mgat1 branching enzyme in human T cells17, where increased expression of IL-7 can either raise or lower N-glycan branching depending on the metabolic supply of UDP-GlcNAc. N- glycan branching enzymes all utilize the common sugar-nucleotide substrate UDP-

GlcNAc, but they do so with declining efficiency such that Mgat4 and Mgat5 are limited for branching by the metabolic production of UDP-GlcNAc. This is due to the Km of Mgat4 and Mgat5 being ~100-200 fold worse than Mgat1 (~0.04 mM). Thus, when UDP-GlcNAc supply is limiting, increasing Mgat1 activity lowers branching by hoarding UDP-GlcNAc away from Mgat4 and Mgat5. Together, this suggests that IL-7 induced increase in Mgat1 activity may synergize with age-dependent increase in UDP-GlcNAc to increase N-glycan branching. Supplementing T cells from mice or humans with GlcNAc raises UDP-GlcNAc levels and N-glycan branching16,22,28. Strikingly, endogenous levels of GlcNAc in human serum markedly increase with age in both females and males (Fig. 2.11A,B). However, this increase in endogenous serum GlcNAc levels only positively correlated with N-glycan branching in female naïve CD4+ T cells but not males (Fig. 2.11C,D), consistent with age- dependent increase in N-glycan branching being greater in females than in males. In addition, co-incubating resting female peripheral blood monocytes (PBMCs) with GlcNAc and IL-7 for 6 or 9 days synergistically increased N-glycan branching in female naïve

CD4+ T cells (Fig. 2.11E,F, 2.12C,D) while only having an additive effect for effector memory CD4+ T cells (Fig. 2.12E,F). Together, our data suggests that elevated IL-7 signaling as a consequence of homeostatic maintenance of the naïve T cell pool in

53 humans synergizes with age-dependent increase in serum GlcNAc to raise N-glycan branching and suppress T cell activity in naïve > memory and female > male.

Discussion

Our current understanding of immune senescence implicates changes within the adaptive immune system, particularly within T cell populations, as the primary determinant of declining immune function with age. Despite our limited mechanistic understanding of immune senescence, the need to demonstrate direct association between immune cell function and clinical outcomes are required to keep pace with the rapidly aging global population. Here we demonstrate that IL-7 driven upregulation of N- glycan branching in female naïve CD4+ T cells contributes to age-dependent immune dysfunction. This is a novel molecular mechanism not previously linked to immune senescence and provides a new avenue for therapeutic targeting. We have demonstrated that inhibiting N-glycan branching either pharmacologically with kifunensine in vitro or via genetic disruption of the branching enzyme Mgat2 rejuvenates immune function in aged

T cells. This extends to rejuvenating immune response in an in vivo model of Salmonella typhimurium infection. Most importantly, our findings point to clear sex discrepancies between old male and female naïve CD4+ T cells. Though previous studies have examined transcriptome changes in aged T cell subsets11,14, this is the first study to our knowledge that specifically examines sex specific changes. Among the relatively few

DEG’s identified between young and old naïve CD4+ T cells, less than 30% of these overlapped between males and females (Table 2.2-2.4), suggesting that immune senescence between sexes is mechanistically different. Immune function differences between males and females are well documented, such as increased risk in women for autoimmune diseases43,44 or increased morbidity and mortality by SARS-CoV-2 among men45. Other aging associated sex differences are also becoming increasingly

55 appreciated. For instance, while immune responses to vaccination are consistently higher in females than males throughout childhood and adulthood, this trend is reversed in aged individuals for the pneumococcal and Td/Tdap vaccines (Klein and Flanagan, 2016). This reversal is consistent with the female specific increase in N-glycan branching observed in this study. Furthermore, while females demonstrate higher antibody titers to influenza vaccination throughout life and in old age, elderly males produce higher affinity antibodies than elderly females46, with the latter more dependent on T cell help. Cumulatively, such sex differences imply that effective interventions of immune senescence in the elderly may require sex-specific strategies.

The mechanism underlying sex-specific increase in N-glycan branching in naïve T cell with age is unclear. Sex chromosome or sex hormone mediated mechanisms are most likely to be involved. Among the IL-7 pathway genes, only the gene encoding IL2rγ, a subunit of the IL-7 receptor, is present on the X-chromosome. However, we did not detect aging associated changes in its gene expression. Sex hormones on the other hand have been previously linked to the IL-7 pathway. In particular, ovariectomy in young female mice was reported to result in higher tissue levels of IL-7, an effect which was

47,48 + reversed by administration of estrogen . Thus, IL-7 driven increases in naïve CD4 T cell branching may be secondary to age associated reduction in estrogen level in females.

By comparison, although sex hormone levels also decrease with age in men, the rate of decrease is much slower49 and may not decrease sufficiently to trigger increased IL-7 production.

Early reports of T cell hypo-reactivity in the elderly used mixed populations. In analyses of distinct T cell subsets, functional defects have persisted in naïve CD4+ T cells

56 but have been brought into question for other subsets50,51. Interestingly, this pattern parallels the magnitude of N-glycan branching observed in this study, with CD4+ > CD8+ and naive > memory. Critically, our findings reveal that increases in N-glycan branching with age are driven by cell extrinsic factors in vivo and quickly reversed when the environment is normalized. Therefore in vitro studies, particularly proliferation studies requiring several days of cell culture, will lead to normalization of branching and will therefore underestimate the true extent of functional impairment. In this regard, in vivo studies and short-term in vitro assays, such as signaling assays in response to TCR stimulation, are likely more accurate measures. However, as aging associated defects have been described in the innate and adaptive arms of the immune system as well as lymphoid tissue architecture, in vivo studies need to be interpreted with caution. To overcome this issue, we used T cell specific branching deficiency (Mgat2fl/fllck-cre mice) in an in vivo model of Salmonella typhimurium infection. Indeed, in aged female mice,

Mgat2 deficiency significantly rejuvenated immune function while leading to no improvement in aged males.

Our previous work revealed that IL7R deficient young mice had higher levels of

N-glycan branching in a mixed population of CD4+ T cells than wild-type littermates17. Our current study showed seemingly contradictory results – in vivo blocking of IL-7 with a neutralizing antibody (M25) resulted in lowered N-glycan branching whereas supplementing with IL-7 increased N-glycan branching. This discrepancy is likely secondary to a number of technical differences between the two studies. Our earlier studies analyzed splenic CD4+ T cells as a whole whereas our study focused on distinguishing between naïve and memory CD4+ T cell populations. Since memory T cells

57 have significantly higher levels of N-glycan branching than naïve T cells, population differences skewed towards memory over naïve in IL7Ra deficient mice36,52 may have accounted for the observed increase in branching. The receptor for thymic stromal lymphopoietin (TSLP) is also absent since it is a heterodimer formed with IL7R as a subunit, consequentially resulting in absent TSLP signaling that may contribute to higher branching in IL7R deficient mice. Since IL7R mutants dramatically effect T cell development in the thymus, higher branching levels may also be a secondary effect of development. Distinguishing and pursuing these effects may shed more light on IL-7’s role and effect on N-glycan branching.

Collectively, our findings provide a novel potential therapeutic target for immune senescence in females. Reversing age-dependent increases in N-glycan branching rejuvenate T cell immunity. Although there are known pharmacological inhibitors, further studies are required to test for toxicity for human use. Using these agents may merit exploration prior to vaccinations or during infection to improve T cell response in elderly females. IL-7 has been a popular non-specific target due to its property to generate T cell receptor diversity53 and promote clonal expansion of mature thymocytes prior to entering the naïve T cell pool54. Treatment of old mice with IL-7 could potentially reverse age- dependent thymic involution55,56 as well as induce homeostatic proliferation to replenish the peripheral naïve T cell pools. Although rejuvenating the naïve T cell population is attractive, our data indicates that this will lead to increased N-glycan branching and reduced functional competence. Thus, IL-7 therapy would be most effective in combination with N-glycan targeting agents that block the IL-7 induced increase in N- glycan branching.

Figure 2.1 N-glycan branching increases with age in mice (A and B) Splenic T cells were analyzed for CD62L and CD44 by flow cytometry (C-F) Flow cytometry analysis of (C and E) female and (D and F) male mouse splenic cells for L-PHA binding, gating on naïve (CD62L+CD44-), central memory (CD62L+CD44+), or effector memory (CD62L-CD44-) as shown in A and B. Normalized geometric mean fluorescence intensity (MFI) is shown. Each aged mouse was normalized to its young control. P-values in (C-F) were determined using two-tailed t-test analysis. Error bars indicate mean ± s.e.m.

Figure 2.2 Lymphocytes from male do not exhibit increased N-glycan branching with age (A) Naïve CD4+ T cells isolated from lymph nodes in young and aged female mice were analyzed for N-glycan branching. (B) Naïve CD4+ T cells from male mice were analyzed for L-PHA binding by flow cytometry. (C and D) Naïve (CD62L+CD44-) CD4+ T cells and CD19+ B cells were directly compared and analyzed for N-glycan branching by L-PHA binding. (E) Thymocytes from female mice were analyzed for L-PHA binding by flow cytometry. Normalized geometric mean fluorescence intensity (MFI) is shown. Each aged mouse was normalized to its young control per experiment. P-values were determined using two-tailed T test. Error bars indicate mean ± s.e.m.

Figure 2.3 Age dependent increase in N-glycan branching suppresses T cell function and differentiation in mice (A-C) Representative flow cytometric analysis of purified splenic CD4+ T cells from the indicated female mouse and strain stimulated by anti-CD3 in the presence or absence of 5µM kifunensine as indicated for (A and B) 24 hours to analyze for activation marker CD69 or (C) 72 hours to assess proliferation by CFSE dilution. (D and E) Flow analysis of purified mouse splenic CD4+ T cells activated with anti-CD3 and anti-CD28 for 4 days with TH17 (TGFβ+IL-6+IL-23) or iTreg (TGFβ) inducing conditions. Data show one experiment representative of at least three independent experiments. Error bars indicate mean ± s.e.m.

Figure 2.4 Regulating N-glycan branching in aged human PBMCs promotes T cell activity and proliferation (A) Flow cytometric analysis of purified memory CD4+ T cells from female mice stimulated by anti-CD3 in the presence or absence of 5µM kifunensine for 24 hours. Data show one experiment representative of at least three independent experiments. Error bars indicate mean ± s.e.m.

Figure 2.5 Genetically regulating N-glycan branching promotes immune response to Salmonella in aged mice Wild Type (WT) and Mgat2-/- mice were pre-treated with streptomycin (0.1ml of a 200mg/ml solution in sterile water) intragastrically prior to inoculation with S. Typhimurium (5x108 colony forming units per mouse). (A-F) Colony forming units (CFU) in Peyer’s patches (A and D), mesenteric lymph nodes (MLN; B and E) and spleen (C and F) were determined at 72 hours after infection in female (A-C) and male (D-F) mice. Normalized proportion of (G) TH17+ and (H) Foxp3+ cells within the gut were detected by flow cytometry. P-values are calculated by unpaired one-tailed t-test with (A) or without (B-F) Welch’s correction. Error bars indicate mean ± s.e.m.

Figure 2.6 Regulating N-glycan branching in aged male mice show minimal effect to Salmonella infection Wild Type (WT) and Mgat2-/- mice were pre-treated with streptomycin (0.1ml of a 200mg/ml solution in sterile water) intragastrically prior to inoculation with S. Typhimurium (5x108 colony forming units per mouse). (A and B) Colony forming units (CFU) in the cecal content (CC) in (A) female and (B) male mice were determined 72 hours after infection. P-values are calculated by unpaired one-tailed t-test. Error bars indicate mean ± s.e.m.

Figure 2.7 Regulating IL-7 signaling reduces N-glycan branching to young phenotype (A) Flow cytometric analysis of freshly isolated naïve CD4+ T cells from the spleen of female mice ex vivo and incubated in complete media for 72 hours after isolation. (B) CD45.2+ naïve CD4+ T cells adoptively transferred into young female CD45.1+ mice analyzed by flow cytometry pre- and post-transfer. (C and D) Flow cytometric analysis of (C) IL7rα and (D) pSTAT5 ex vivo in female mice. (E and F) L-PHA flow cytometry of naïve CD4+ T cells from female mice treated with or without rhIL-7 (50ng/ml). (G) Mice received intraperitoneal injections of either isotype control (1.5µg) or rhIL-7/M25 on days 1, 3, and 5. On day 7, spleen were harvested and analyzed by LPHA flow cytometry. (H) Mice received intraperitoneal injections of either isotype control (1.5mg) or anti- mouse/human IL-7 (M25, 1.5mg) antibody 3 times a week for two weeks and had both blood drawn from the tail-tip for weekly assessment of N-glycan branching on naïve CD4+ T cells. (I) On day 14, spleens were harvested and LPHA was determined by flow cytometry. (J and K) Flow cytometric analysis of (J) IL7rα and (K) pSTAT5 of mice treated with either isotype control (1.5mg) or anti-mouse/human IL-7 (M25, 1.5mg) antibody 3 times a week for four weeks. P-values in (A) were determined using one-sided Mann- Whitney test. Error bars indicate mean ± s.e.m.

Figure 2.8 IL-7 signaling pathway increases with age to promote N-glycan branching (A) Representative flow cytometry plots of CD45.2+ donor cells in CD45.1+ recipient mice. (B) Analysis of sorted cells prior to RNA isolation and library prep for RNA-seq (C) Number of differentially expressed genes (DEGs) between young and aged naïve CD4+ T cells in female, male, or both. (D) qPCR of critical Golgi branching enzymes in female mice. (E) Mice received intraperitoneal injections of either isotype control (1.5mg) or anti- mouse/human IL-7 (M25, 1.5mg) antibody 3 times a week for two weeks and had their spleen harvested and analyzed for IL7rα. (F) Mice received intraperitoneal injections of either isotype control (1.5mg) or anti-mouse/human IL-7 (M25, 1.5mg) antibody 3 times a week for four weeks and had blood drawn from the tail-tip for weekly assessment of N- glycan branching on naïve CD4+ T cells. (G) On day 28, spleens were harvested and LPHA was determined by flow cytometry. P-values are calculated by unpaired one-tailed t-test. Error bars indicate mean ± s.e.m.

Figure 2.9 N-glycan branching increases with age in humans (A-D) Human PBMCs from (A, C and D) females and (B) males were analyzed for L-PHA by flow cytometry in (A and B) CD4+ T cells, (C) CD8+ T cells, and CD19+ (D) B cells. (E) CD4+ T cells were further gated into naïve (CD45RA+CD45RO-) and memory (CD45RA-CD45RO+). (F) N-glycan branching was assessed for naïve and memory CD4+ T cells. Human data are from PBMCs isolated from healthy individuals as indicated in materials and methods. P-values were determined using linear regression analysis. Error bars indicate mean ± s.e.m.

Figure 2.10 Age-dependent increase in N-glycan branching also suppresses T cell activity in humans (A-F) Human PBMCs from males were analyzed for LPHA by flow cytometry with further gating onto (A) CD8+ T cells, (B) CD19+ B cells, (C) naïve (CD45RA+CD45RO-) and (D) memory (CD45RA-CD45RO+) CD4+ T cells. (E and F) Total CD4+ T cells stained from (E) females and (F) males under the age of 65 were analyzed by LPHA flow cytometry. Human data are from PBMCs isolated from healthy individuals as indicated in materials and methods. P-values were determined using linear regression analysis. Error bars indicate mean ± s.e.m.

Figure 2.11 IL-7 synergizes with age-dependent increase in endogenous N- acetylglucosamine to increase N-glycan branching in humans (A and B) Serum from humans were analyzed for levels of GlcNAc by LC-MS/MS. (C and D) PBMC’s taken at the same time were analyzed by LPHA flow cytometry of naïve CD4+ T cells from (C) female and (D) male donors. (E and F) PBMCs from a 28 years old female Caucasian were treated with or without GlcNAc (40mM) and/or rhIL-7 (50ng/ml) then analyzed by LPHA flow cytometry. P-values were determined using (A-D) linear regression or (E-F) unpaired one-tailed t-test analysis. Error bars indicate mean ± s.e.m.

Figure 2.12 Inhibiting N-glycosylation in human cells reverse age-induced suppression of T cell activity by N-glycan branching (A and B) Flow cytometric analysis of female human PBMCs stimulated by anti-CD3 in the presence or absence of 5µM kifunensine as indicated for (A) 24 hours to analyze for activation marker CD69 or (B) 72 hours to assess proliferation by CFSE dilution. (C-F) PBMCs from a 28 years old female Caucasian were treated with or without GlcNAc (40mM) and/or rhIL-7 (50ng/ml) for (C-E) 6 days or (F) 9 days then analyzed by LPHA flow cytometry. Data show one experiment representative of at least three independent experiments. Error bars indicate mean ± s.e.m.

Table 2.1 Female and Male IL7 Signaling Pathway DEGs Female Male Log2 Fold Log2 Fold Gene Symbol Gene Description Adj p Value Adj p Value Change Change Suppressor of cytokine Socs3 4.49E-21 -2.5633 6.84E-01 -0.8798 signaling 3

Jak3 Janus kinase 3 6.67E-15 -1.4272 1.20E-01 -0.5597

IL7ra Interleukin 7 receptor alpha 1.80E-02 0.6727 7.42E-01 0.2146

Signal transducer and Stat5a 6.36E-01 -0.3568 9.27E-01 -0.1486 activator of transcription 5A

Jak1 Janus kinase 1 6.62E-01 0.4007 9.77E-01 -0.0647

Il2rg Interleukin 2 receptor gamma 7.52E-01 -0.2393 9.49E-01 -0.0762

Signal transducer and Stat5b 9.93E-01 -0.0097 8.54E-01 0.1410 activator of transcription 5B

Table 2.2 Female Only Young vs Old Naïve DEGs Log2 Fold Gene Symbol Gene Description Adj. p value Change 1700066B19Rik RIKEN cDNA 1700066B19 gene 1.53E-02 -2.0579 5830416I19Rik RIKEN cDNA 5830416l19 gene 1.66E-07 -6.8686 Abi3 ABI gene family, member 3 1.22E-02 -0.8102 Acot7 acyl-CoA thioesterase 7 1.70E-03 -1.1811 Adcy7 Adenylate cyclase 7 2.71E-02 0.6169 Anxa2 Annexin A2 4.80E-02 -1.1599 Apobec3 Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 6.91E-03 -0.8127 B430306N03Rik RIKEN cDNA B430306N03 gene 2.10E-02 -1.2834 Bcl3 B cell leukemia/lymphoma 3 1.26E-19 -2.2804 Bhlhe40 Basic helix-loop-helix family, member e40 1.35E-02 -2.3633 Camk2b Calcium/Calmodulin-dependent protein kinase II, beta 1.07E-10 3.0108 Ccr5 Chemokine (C-C motif) receptor 5 1.20E-02 -3.7143 Cd55 CD55 molecule, decay accelerating factor for complement 8.26E-03 0.8636 Cd74 CD74 antigen 2.62E-22 -2.9560 Cdk5rap1 CDK5 regulatory subunit associated protein 1 9.81E-08 1.5333 Cdkn2d Cyclin dependent kinase inhibitor 2D 1.96E-03 -0.7664 Chchd10 Coiled-coil-helix-coiled-coil-helix domain containing 10 1.23E-03 -1.1373 Cirbp Cold inducible RNA binding protein 9.58E-03 -1.0224 Ckb Creatine kinase, brain 2.88E-03 -0.8103 Cldn10 Claudin 10 3.42E-07 -2.2617 Cnn3 Calponin 3, acidic 1.50E-02 -0.6822 Crim1 Cysteine rich transmembrane BMP regulator 1 (chordin like) 6.11E-03 0.9050 Ctsa Cathepsin A 1.02E-04 -0.8668 Cttn Cortactin 3.99E-02 -3.5398 Cxcr1 Chemokine (C-X-C motif) receptor 1 4.26E-04 -2.1348 Cxcr3 Cheomkine (C-X-C motif) receptor 3 2.03E-02 -2.3439 Cxcr5 Chemokine (C-X-C motif) receptor 5 3.85E-07 -2.7931 Cysltr2 Cysteinyl leukotriene receptor 2 1.96E-03 -4.5641 Dse Dermatan sulfate epimerase 5.44E-04 1.1449 E2f2 E2F transcription factor 2 5.07E-03 -0.7820 Egln3 egl-9 family hypoxia-inducible factor 3 3.30E-02 -1.9053 Elovl6 ELOVL fatty acid elongase 6 2.60E-03 -1.1859 Elovl7 ELOVL fatty acid elongase 7 4.60E-02 1.0645 Eomes Eomesodermin 1.25E-02 -1.7488 Erdr1 Erythroid differentiation regulator 1 4.63E-03 -1.4042 Eri2 Exoribonuclease 2 1.72E-02 0.9227 Etv6 ETS variant transcription factor 6 1.57E-03 -1.2455 Fam212b Inka box actin regulator 2 3.31E-02 1.2568 Fasl Fas ligand (TNF superfamily, member 6) 3.09E-02 1.2776 Gadd45g Growth arrest and DNA-damage-inducible 45 gamma 1.07E-06 -3.4287 Gatsl3 Cytosolic arginine sensor for mTORC1 subunit 1 6.25E-03 -0.9378 Gbp3 Guanylate binding protein 3 3.53E-02 -0.9029 Gm4956 Predicted gene 4956 3.91E-06 -2.8674 Gngt2 G protein subunit gamma transducing 2 1.22E-02 -1.1063 Gpd2 Glycerol phosphate dehydrogenase 2, mitochondrial 2.25E-06 1.5306 H2-Aa Histocompatibility 2, class II antigen A, alpha 2.88E-03 -2.8507 H2-Ab1 Histocompatibility 2, class II antigen A, beta 1 9.99E-03 -3.4321 H2-Eb1 Histocompatibility 2, class II antigen E beta 3.16E-02 -2.5872 H2-Oa Histocompatibility 2, O region alpha locus 2.69E-02 -1.1386 Hspa1a Heat shock protein 1A 1.17E-03 2.5859 Hsph1 Heat shock 105kDa/110kDa protein 1 1.42E-04 1.1183 Igfbp4 Insulin-like growth factor binding protein 4 1.09E-04 -0.9894 Iigp1 Interferon inducible GTPase 1 2.02E-09 -1.4493 Ikzf4 IKAROS family zinc finger 4 2.61E-02 -1.2427 Il18bp Interleukin 18 binding protein 2.48E-03 -2.4191 Il7r Interleukin 7 receptor 1.76E-02 0.6727 Irf8 Interferon regulatory factor 8 1.07E-03 -1.3325 Itga6 Integrin alpha 6 9.12E-03 0.9563 Itih5 Inter-alpha (globulin) inhibitor H5 1.49E-03 -1.3769 Itk IL2 inducible T cell kinase 3.53E-02 0.6286 Jak3 Janus kinase 3 6.67E-15 -1.4272 Jund JunD proto-oncogene 3.43E-02 -0.5916 Jup Junction plakoglobin 3.53E-02 -1.2538 Lag3 Lymphocyte-activation gene 3 4.71E-07 -3.7146 Mbnl3 Muscleblind like splicing factor 3 2.89E-02 0.6476 Moxd1 Monooxygenase, DBH-like 1 2.15E-02 -5.9660

Myb Myeloblastosis oncogene 3.30E-02 -1.1794 Nebl Nebulette 2.61E-02 3.3010 Nfkbia NFKB inhibitor alpha 4.16E-02 -0.7300 Nkg7 Natural killer cell group 7 sequence 2.29E-11 -4.4538 Nr4a1 Nuclear receptor subfamily 4, group A, member 1 4.94E-02 -0.7616 Nrn1 Neuritin 1 6.54E-03 -1.5580 Ntrk3 Neurotrophic tyrosine kinase, receptor, type 3 3.19E-02 0.9271 Pcdhgc3 Protocadherin gamma subfamily C, 3 4.63E-03 -3.2355 Pdcd1 Programmed cell death 1 (PD-1) 3.09E-02 -1.0694 Pde2a Phosphodiesterase 2A, cGMP-stimulated 4.92E-02 0.6572 Pde3b Phosphodiesterase 3B, cGMP-inhibited 1.08E-08 1.1025 Pdlim4 PDZ and LIM domain 4 1.07E-06 1.4591 Pim2 Proviral integration site 2 1.07E-03 -0.7646 Plek Pleckstrin 1.35E-02 -2.9786 Ptms Parathymosin 3.42E-03 -0.7579 Relb Avian reticuloendotheliosis viral (v-rel) oncogene related B 3.44E-02 -0.6937 Rgcc Regulator of cell cycle 2.25E-02 0.7478 Rragd Ras-related GTP binding D 3.09E-02 -1.4669 S100a4 S100 calcium binding protein A4 1.89E-02 -2.7483 S100a6 S100 calcium binding protein A6 (calcyclin) 3.09E-02 -2.5779 Sbno2 Strawberry Notch 2 6.53E-06 -1.1509 Scand1 SCAN domain-containing 1 1.96E-02 -0.9716 Sdhaf1 Succinate dehydrogenase complex assembly factor 1 1.09E-02 -0.9208 Sema4f Semaphorin 4F 9.82E-04 0.9065 Sema7a Semaphorin 7A 3.25E-02 -4.1315 Sgk1 Serum/glucocorticoid regulated kinase 1 1.96E-03 -0.7464 Skap2 src family associated phosphoprotein 2 3.78E-05 -1.3032 Sntb1 Syntrophin, basic 1 1.10E-03 1.4221 Sntb2 Syntrophin, basic 2 1.62E-03 1.9643 Socs1 Suppressor of cytokine signaling 1 6.00E-06 -1.4015 Socs3 Suppressor of cytokine signaling 3 4.49E-21 -2.5633 Ssbp4 Single stranded DNA binding protein 4 1.23E-03 -0.8345 Susd3 Sushi domain containing 3 3.74E-02 -0.7478 Synpo Synaptopodin 3.30E-02 -3.2933 Tgm2 Transglutaminase 2, C polypeptide 4.66E-02 -5.2961 Tgtp1 T cell specific GTPase 1 1.96E-02 -0.7238 Tha1 Threonine aldolase 1 2.42E-05 -1.7877 Tlr12 Toll-like receptor 12 1.35E-02 -1.0229 Tm6sf1 Transmembrane 6 superfamily member 1 1.22E-02 -1.1883 Tnfaip8 Tumor necrosis factor, alpha-induced protein 8 1.84E-03 0.7255 Tnfrsf19 Tumor necrosis factor receptor superfamily, member 19 4.16E-02 2.6928 Tnfrsf4 Tumor necrosis factor receptor superfamily, member 4 7.79E-03 -1.1326 Tnfrsf9 Tumor necrosis factor receptor superfamily, member 9 8.79E-05 -1.3290 Tppp Tubulin polymerization promoting protein 2.25E-02 5.6820 Tspan3 Tetraspanin 3 4.60E-02 -0.6839 Ttc28 Tetratricopeptide repeat domain 28 2.61E-02 1.1526 Tubb2b Tubulin, beta 2B class IIB 8.60E-06 -1.7083 Vamp2 Vesicle-associated membrane protein 2 3.53E-02 -0.6872

Table 2.3 Male Only Young vs Old Naïve DEGs Log2 Fold Gene Symbol Gene Description Adj. p Value Change 1110032F04Rik Riken cDNA 1110032F04 gene 1.46E-05 1.1845 2310022B05Rik Riken cDNA 2310022B05 gene 4.62E-02 0.8614 2410006H16Rik Riken cDNA 2410006H16 gene 3.56E-03 -0.8239 5830416I19Rik Riken cDNA 5830416I19 gene 2.14E-12 -5.1989 Abca1 ATP-binding cassette, sub-family A (ABC1), member 1 1.84E-03 1.2653 Abcb9 ATP-binding cassette, sub-family B (MDR/TAP), member 9 3.35E-02 0.5156 Acsf2 Acyl-CoA synthetase family member 2 1.47E-02 0.8510 Ahnak AHNAK nucleoprotein (desmoyokin) 4.87E-02 -1.3980 Aim2 Absent in melanoma 2 8.23E-03 -1.0271 Aqp3 Aquaporin 3 3.35E-02 0.8785 Arsb Arylsulfatase B 1.02E-02 0.5862 Atp5o ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit 3.09E-02 -0.3667 B4galt5 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 5 3.18E-02 0.6390 Bach2 BTB and CNC homology, basic leucine zipper transcription factor 2 2.23E-02 0.5546 Cd52 CD52 antigen 4.04E-02 -0.3856 Cd7 CD7 antigen 3.79E-03 0.8743 Cd72 CD72 antigen 3.17E-03 -1.1841 Cmpk2 Cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial 3.35E-02 0.6314 Cox7a2l Cytochrome c oxidase subunit 7A2 like 3.32E-02 -0.4024 Cst7 Cystatin F (leukocystatin) 2.01E-07 -1.4238 Ctdsp2 CTD small phosphatase 2 2.37E-02 0.4188 Cyp4v3 Cytochrome P450, family 4, subfamily v, polypeptide 3 3.56E-03 -0.8192 Cyp51 Cytochrome P450, family 51 1.42E-03 -0.7610 Dbi Diazepam binding inhibitor 4.63E-02 -0.7428 Dnajc15 DnaJ heat shock protein family (Hsp40) member C15 2.83E-02 -0.5734 Dos Calcium channel, beta subunit associated regulatory protein 3.06E-02 1.1173 Dusp2 Dual specificity phosphatase 2 1.26E-02 0.6474 Eef1a1 Eukaryotic translation elongation factor 1 alpha 1 3.85E-02 -0.3604 Eef1d Eukaryotic translation elongation factor 1 delta 4.04E-02 -0.4543 Eef1g Eukaryotic translation elongation factor 1 gamma 2.78E-02 -0.3684 Eif3k Eukaryotic translation initiation factor 3, subunit K 1.35E-02 -0.3968 Eif3m Eukaryotic translation initiation factor 3, subunit M 3.35E-02 -0.4399 Fam46a Family with sequence similarity 46, member A 8.49E-03 -0.4775 Fau FBR-MuSV ubiquitously expressed (fox derived) 4.69E-02 -0.4788 Fdft1 Farnesyl diphosphate farnesyl transferase 1 2.58E-02 -0.6958 Fdps Farnesyl diphosphate synthetase 1.16E-02 -0.6462 Gapdh Glyceraldehyde-3-phosphate dehydrogenase 4.55E-02 -0.3065 Ggta1 Glycoprotein galactosyltransferase alpha 1, 3 3.44E-02 -0.6031 Gltscr2 NOP53 ribosome biogenesis factor 4.77E-02 -0.4197 Gm128 Predicted gene 128 4.43E-02 -5.2156 Gpc1 Glypican 1 1.48E-03 -1.3638 Gpr34 G protein-coupled receptor 34 2.48E-03 -1.1009 Gpr68 G protein-coupled receptor 68 1.51E-03 -1.4243 Gpx4 Glutathione peroxidase 4 7.45E-03 -0.4584 Gucy1a3 Guanylate cyclase 1, soluble, alpha 1 5.11E-03 -1.7567 H2-Q10 Histocompatibility 2, Q region locus 10 2.23E-02 -2.7733 Hmgcs1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 1.53E-02 -0.6002 Hnrnpdl Heterogeneous nuclear ribonucleoprotein D-like 3.06E-02 0.6533 Hs3st3b1 Heparan sulfate (glucosamine) 3-O-sulfotransferase 3B1 3.91E-02 0.5637 Hsp90ab1 Heat shock protein 90 alpha (cytosolic), class B member 1 1.15E-03 -0.6139 Idi1 Isopentenyl-diphosphate delta isomerase 1.02E-02 -0.7977 Ifi27l2a Interferon, alpha-inducible protein 27 like 2A 3.43E-06 0.7556 Ifit1 Interferon-induced protein with tetratricopeptide repeats 1 1.47E-04 1.0389 Ifit3 Interferon-induced protein with tetratricopeptide repeats 3 1.29E-04 1.1835 Igbp1 Immunoglobulin (CD79A) binding protein 1 4.04E-02 -0.4280 Igf2bp3 Insulin-like growth factor 2 mRNA binding protein 3 1.44E-02 5.8260 Impdh2 Inosine monophosphate dehydrogenase 2 1.62E-03 -0.4951 Irf2bp2 Interferon regulatory factor 2 binding protein 2 3.51E-02 0.5202 Irf7 Interferon regulatory factor 7 6.19E-03 0.6545 Klf2 Kruppel-like factor 2 (lung) 2.30E-02 0.4169 Klf3 Kruppel-like factor 3 (basic) 1.10E-02 0.5150 Klhdc3 Kelch domain containing 3 4.15E-02 0.4574 Ldlr Low density lipoprotein receptor 4.48E-03 -0.8302 Lpxn Leupaxin 1.35E-02 -0.5700 Ly6k Lymphocyte antigen 6 complex, locus K 1.26E-03 -1.7027 Mapk11 Mitogen-activated protein kinase 11 4.62E-02 -0.7311

Mex3b Mex3 RNA binding family member B 1.12E-02 0.9307 Mex3c Mex3 RNA binding family member C 1.00E-02 0.6569 Mrps16 Mitochondrial ribosomal protein S16 3.16E-02 -0.5350 Mthfd2 Methylenetetrahydrofolate dehydrogenase 4.63E-02 -0.4824 Myeov2 Myeloma overexpressed 2 homolog 2.27E-02 -0.5461 Mylip Myosin regulatory light chain interacting protein 4.87E-02 0.4050 Myo6 Myosin VI 1.45E-05 -1.0580 Nme2 NME/NM23 nucleoside diphosphate kinase 2 3.56E-03 -0.5086 Npm1 Nucleophosmin 1 4.18E-02 -0.3434 Nsa2 NSA2 ribosome biogenesis homolog 6.32E-03 -0.4449 Oas2 2'-5' oligoadenylate synthetase 2 4.87E-02 2.0424 Oasl2 2'-5' oligoadenylate synthetase-like 2 7.14E-04 1.3155 Pag1 Phosphoprotein associated with glycosphingolipid microdomains 1 1.28E-02 0.4532 Pfdn5 Prefoldin 5 1.89E-03 -0.5263 Phgdh 3-phosphoglycerate dehydrogenaseprovided 2.78E-02 -0.4446 Pid1 Phosphotyrosine interaction domain containing 1 1.23E-03 -2.1133 Plagl2 Pleiomorphic adenoma gene-like 2 2.30E-02 0.5996 Plp2 Proteolipid protein 2 1.89E-03 -0.6411 Prf1 Perforin 1 (pore forming protein) 4.97E-03 -0.5866 Pros1 Protein S (alpha) 1.34E-02 -1.8707 Ptgfrn Prostaglandin F2 receptor negative regulator 1.10E-02 0.9535 Ptpn6 Protein tyrosine phosphatase, non-receptor type 6 4.39E-02 -0.3542 Rab11fip4 RAB11 family interacting protein 4 (class II) 2.23E-02 0.7785 Ranbp10 RAN binding protein 10 8.32E-03 0.5987 Rasal1 RAS protein activator like 1 (GAP1 like) 4.64E-03 1.4741 Rnf122 Ring finger protein 122 2.48E-03 -0.8446 Rpl10 Ribosomal protein L10 1.90E-03 -0.4722 Rpl11 Ribosomal protein L11 1.83E-02 -0.4325 Rpl13a Ribosomal protein L13A 2.00E-02 -0.4755 Rpl14 Ribosomal protein L14 6.75E-03 -0.4597 Rpl17 Ribosomal protein L17 4.48E-03 -0.4799 Rpl3 Ribosomal protein L3 1.88E-02 -0.4605 Rpl32 Ribosomal protein L32 2.39E-02 -0.4525 Rpl35 Ribosomal protein L35 3.44E-02 -0.4375 Rpl35a Ribosomal protein L35A 4.87E-02 -0.4262 Rpl36a Ribosomal protein L36A 2.00E-02 -0.5360 Rpl37a Ribosomal protein L37a 3.09E-02 -0.4574 Rpl38 Ribosomal protein L38 2.86E-02 -0.5145 Rpl39 Ribosomal protein L39 2.78E-02 -0.5456 Rpl4 Ribosomal protein L4 2.17E-02 -0.3358 Rpl5 Ribosomal protein L5 7.06E-03 -0.4216 Rpl6 Ribosomal protein L6 2.39E-02 -0.3729 Rpl7a Ribosomal protein L7A 4.80E-02 -0.3587 Rps13 Ribosomal protein S13 4.43E-02 -0.4188 Rps15a Ribosomal protein S15A 4.15E-02 -0.4271 Rps20 Ribosomal protein S20 3.10E-02 -0.4667 Rps21 Ribosomal protein S21 3.20E-02 -0.5277 Rps29 Ribosomal protein S29 1.21E-02 -0.6317 Rps3 Ribosomal protein S3 2.95E-02 -0.4492 Rps6 Ribosomal protein S6 1.90E-02 -0.4244 Rps8 Ribosomal protein S8 4.69E-02 -0.3784 Rps9 Ribosomal protein S9 1.68E-02 -0.5002 Rsad2 Radical S-adenosyl methionine domain containing 2 1.19E-03 1.5841 Rtp4 Receptor transporter protein 4 3.30E-05 0.8465 Sdc3 Syndecan 3 8.53E-03 1.0743 Sec61g SEC61, gamma subunit 3.09E-02 -0.5096 Sema4a Semaphorin 4A 2.30E-02 -0.4651 Skil SKI-like 4.87E-02 0.3824 Slc25a39 Solute carrier family 25, member 39provided 2.79E-02 0.5353 Slc35g1 Solute carrier family 35, member G1 1.90E-03 0.8306 Snrpg Small nuclear ribonucleoprotein polypeptide G 1.67E-02 -0.4938 Sp140 Sp140 nuclear body protein 6.75E-03 -0.6385 Sqle Squalene epoxidase 2.06E-03 -0.9184 Srpk2 Serine/arginine-rich protein specific kinase 2 1.12E-02 0.5446 Sugt1 SGT1, suppressor of G2 allele of SKP1 (S. cerevisiae) 4.25E-03 -0.5729 Tapbpl TAP binding protein-like 1.42E-03 -0.6756 Tbc1d30 TBC1 domain family, member 30 4.64E-03 -1.0111 Tet3 Tet methylcytosine dioxygenase 3 6.75E-03 0.7656 Tmem184b Transmembrane protein 184b 4.14E-02 0.6102 Tnfrsf26 Tumor necrosis factor receptor superfamily, member 26 3.43E-02 0.5553

Tnfsf8 Tumor necrosis factor (ligand) superfamily, member 8 1.02E-03 -0.7095 Tob1 Transducer of ErbB-2.1 1.02E-03 0.8293 Tomm7 Translocase of outer mitochondrial membrane 7 1.65E-03 -0.7633 Traf1 TNF receptor-associated factor 1 1.66E-02 -0.6020 Trim25 Tripartite motif-containing 25 3.56E-03 0.5644 Trim59 Tripartite motif-containing 59 4.14E-02 0.4341 Tspan31 Tetraspanin 31 1.82E-03 -0.8866 Unc5a Unc-5 netrin receptor A 2.99E-02 -1.9817 Uqcrh Ubiquinol-cytochrome c reductase hinge protein 4.87E-02 -0.4087 Use1 Unconventional SNARE in the ER 1 homolog (S. cerevisiae) 3.28E-02 -0.4644 Usp18 Ubiquitin specific peptidase 18 4.64E-03 0.7152 Zfp36l2 Zinc finger protein 36, C3H type-like 2 3.82E-03 0.5878

Table 2.4 Female and Male Overlapping DEGs Female Male Log2 Fold Log2 Fold Gene Symbol Adj p Value Adj p Value Change Change 1500012F01Rik 4.94E-03 -1.0524 1.46E-05 -1.1708 2410006H16Rik 4.38E-04 -1.0788 1.07E-03 -1.1059 Adss 3.58E-04 -0.7469 5.06E-08 -0.6860 Aldh1l1 9.61E-03 -1.6002 1.79E-05 -2.6125 Apol10b 1.57E-08 -3.6822 9.09E-16 -4.3784 Appl2 3.79E-03 1.1352 1.83E-02 1.1032 Arhgap29 7.84E-06 1.5590 9.05E-03 0.6801 AW112010 5.79E-06 -1.0852 1.69E-11 -1.1010 Bmpr1a 4.49E-21 -3.8931 2.00E-18 -3.1579 Cacnb3 5.86E-08 -2.8478 4.63E-02 -1.4560 Casp1 3.15E-11 -3.0164 2.69E-03 -3.5607 Casp4 3.42E-07 -3.5999 1.43E-09 -3.4210 Ccl5 4.49E-21 -5.4775 9.36E-03 -2.8902 Cd200r4 1.80E-06 -2.6842 2.36E-06 -2.0723 Cd86 2.09E-03 1.7294 2.10E-02 1.5134 Dtx1 1.04E-02 0.7035 8.73E-06 0.8036 Enc1 1.49E-03 0.8184 1.53E-03 0.6091 Fbxo17 1.02E-05 -2.3884 1.73E-03 -1.7169 Gas5 2.71E-02 -0.7366 2.40E-06 -0.9060 Gbp2 2.16E-02 -0.7099 3.95E-03 -0.6231 Gm4841 1.02E-04 -5.7597 8.12E-04 -3.6705 Gm4951 1.49E-04 -6.4540 1.59E-04 -4.3977 Gm7120 1.22E-02 -1.2963 4.64E-03 -1.3632 Gm7609 3.42E-07 -2.9338 1.66E-12 -4.0232 Gzmk 1.08E-08 -8.7232 2.86E-02 -4.4424 H2-DMa 2.15E-04 -0.9710 1.12E-05 -0.9216 Icam1 1.10E-03 -1.0113 1.81E-02 -0.7490 Ildr1 2.07E-02 2.4758 1.67E-03 2.6255 Insl6 2.83E-04 -1.3229 3.84E-05 -1.3871 Klrd1 1.10E-03 1.4582 3.84E-05 1.4034 Mrpl52 4.94E-02 -0.7541 3.80E-02 -0.5990 Nacc2 1.71E-04 0.9090 1.38E-08 1.1583 Ndrg3 1.81E-05 -0.8692 4.14E-02 -0.4223 Nnt 1.17E-08 1.3178 3.40E-12 1.2261 Pdcd4 3.52E-03 0.7233 1.10E-02 0.4678 Rab4a 1.57E-14 3.8931 5.28E-15 4.6030 Raver2 1.66E-02 -1.0172 3.56E-03 -1.0146 Rell1 2.05E-05 -1.7039 3.14E-10 -2.1798 Sox4 4.81E-02 -0.7521 4.87E-02 0.8960 Synj2 3.52E-03 -1.4869 5.04E-11 -1.6958 Syt11 5.47E-06 -1.0334 2.95E-02 -0.7932 Tdrp 1.98E-04 0.7991 1.49E-02 0.5720 Tspan9 3.42E-07 1.8474 7.99E-13 2.4207 Wdfy1 3.06E-16 3.4306 1.86E-17 2.7677

Table 2.5 Female Glycosylation Genes and Other DEGs Log2 Fold Gene Symbol Gene Description Adj. p value Change Adpgk ADP-dependent glucokinase 0.2667 0.4553 B3gnt1 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 1 0.9492 -0.0719 B3gnt2 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2 0.9933 -0.0089 B3gnt3 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 3 0.1635 -1.1030 B3gnt4 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 4 NA NA B3gnt5 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 5 0.8926 0.2225 B3gnt6 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 6 NA NA B3gnt7 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 7 NA NA B3gnt8 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 8 0.8538 -1.3941 B3gnt9 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 9, 0.7621 -0.8225 B3gntl1 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase-like 1 0.7954 -0.4591 B4galnt4 Beta-1,4-N-acetyl-galactosaminyl transferase 4 NA -2.1628 B4galt1 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 1 0.8171 0.1699 B4galt2 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 2 NA NA B4galt3 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 3 0.9838 -0.0304 B4galt4 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 4 NA 0.6647 B4galt5 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 5 0.8672 0.1776 B4galt6 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 6 NA NA B4galt7 Beta1,4-galactosyltransferase 0.9916 0.0163 Eno1 Enolase 1, alpha-non-neuron NA NA Eno1b Enolase 1B, retrotransposed 0.9874 -0.0574 Eno2 Enolase 2 0.9951 -0.0548 Eno3 Enolase 3 0.9750 -0.0723 Eno4 Enolase 4 NA NA G6pdx Glucose 6-phosphate dehydrogenase X-linked 0.8639 -0.1468 Gale Galactose-4-epimerase, UDP 0.9290 -0.1841 Gck Glucokinase NA NA Gfpt1 Glutamine fructose-6-phosphate transaminase 1 0.8732 0.1748 Gfpt2 Glutamine fructose-6-phosphate transaminase 2 NA -1.7757 Gls Glutaminase 0.7603 0.2730 Glud1 Glutamate dehydrogenase 1 0.8985 0.0978 Gne UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase 0.6054 0.3886 Gnpda1 Glucosamine-6-phosphate deaminase 1 0.8257 -0.1663 Gnpda2 Glucosamine-6-phosphate deaminase 2 0.9777 -0.0309 Gnpnat1 Glucosamine-phosphate N-acetyltransferase 1 0.7259 -0.2546 Got1 Glutamic-oxaloacetic transaminase 1 0.9740 -0.0327 Got1l1 Glutamic-oxaloacetic transaminase 1 like 1 NA NA Got2 Glutamic-ocaloacetic transaminase 2 0.6621 -0.2742 Gpi1 Glucose phosphate isomerase 1 0.5984 -0.3103 Gpt Glutamic pyruvic transaminase, soluble 0.8542 -0.2380 Gpt2 Glutamic pyruvic transaminase (alaline aminotransferase) 2 0.9966 0.0295 Hk1 Hexokinase 1 0.9094 0.1194 Hk2 Hexokinase 2 NA 1.1863 Hk3 Hexokinase 3 NA 1.6616 Ldha Lactate dehydrogenase A 0.8161 -0.1629 Man1a Mannosidase 1, alpha 0.8152 0.2135 Man1a2 Mannosidase, alpha, class 1A, member 2 0.7477 0.2740 Man1b1 Mannosidase, alpha, class 1B, member 1 0.7946 0.1982 Man1c1 Mannosidase, alpha, class 1C, member 1 0.8367 0.3325 Man2a1 Mannosidase 2, alpha 1 0.6904 0.4323 Man2a2 Mannosidase 2, alpha 2 0.6098 0.4007 Man2b1 Mannosidase 2, alpha B1 0.9458 -0.0666 Man2b2 Mannosidase 2, alpha B2 0.9094 -0.1241 Man2c1 Mannosidase, alpha, class 2C, member 1 0.9147 0.1061 Mgat1 Mannoside acetylglucosaminyltransferase 1 0.9951 -0.0066 Mgat2 Mannoside acetylglucosaminyltransferase 2 0.9848 0.0200 Mgat3 Mannoside acetylglucosaminyltransferase 3 NA NA Mgat4a Mannoside acetylglucosaminyltransferase 4, isoenzyme A 0.9886 0.0181 Mgat4b Mannoside acetylglucosaminyltransferase 4, isoenzyme B 0.8412 -0.4425 Alpha-1,3-mannosyl-glycoprotein beta-1,4-N- Mgat4c NA acetylglucosaminyltransferase, isozyme C (putative) NA Mgat5 Mannoside acetylglucosaminyltransferase 5 0.9951 0.0155 Mgat5b Mannoside acetylglucosaminyltransferase 5, isoenzyme B NA NA Nagk N-acetylglucosamine kinase 0.9725 -0.0562 Nat9 N-acetyltransferase 9 (GCN5-related, putative) 0.8148 -0.3844 Pfkfb1 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 0.9926 -0.0792

Pfkfb2 6-phosphofructo-2-kinase/fructore-2,6-biphosphatase 2 0.9352 0.1375 Pfkfb3 6-phosphofructo-2-kinase/fructore-2,6-biphosphatase 3 0.8717 0.1664 Pfkfb4 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 0.9886 0.0325 Pfkl Phosphofructokinase, liver, B-type 0.8665 0.1334 Pfkm Phosphofructokinase, muscle 0.8862 -0.1830 Pfkp Phosphofructokinase, platlet 0.9872 0.0170 Pgd 6-phosphogluconate dehydrogenase 0.8995 0.1036 Pgls 6-phosphogluconolactonase 0.1747 -0.6230 Pgm1 Phosphoglucomutase 1 0.6213 -0.3442 Pgm2 Phosphoglucomutase 2 0.5783 -0.3919 Pgm2l1 Phosphoglucomutase 2-like 1 0.5350 0.5911 Pgm3 Phosphoglucomutase 3 0.9454 0.0952 Pgm5 Phosphoglucomutase 5 NA NA Pklr Pyruvate kinase liver and red blood cell NA NA Pkm Pyruvate kinase, muscle 0.7581 -0.1902 Rpe Ribulase-5-phosphase-3-epimerase 0.8242 0.1568 Rpia Ribose 5-phosphate isomerase A 0.9838 0.0237 Slc1a5 Solute carrier family 1 (neutral amino acid transporter), member 5 0.6652 -0.3384 Slc2a1 Solute carrier family 2 (facilitated glucose transporter), member 1 0.9792 -0.0278 Slc2a2 Solute carrier family 2 (facilitated glucose transporter), member 2 NA -0.8863 Slc2a4 Solute carrier family 2 (facilitated glucose transporter), member 4 NA NA Slc2a4rg-ps Slc2a4 regulator, pseudogene 0.7881 -0.5256 Slc35a3 Solute carrier family 35 (UDP-GlcNAc transporter), member 3 0.8936 0.1578 Slc35d1 Solute carrier family 35, member D1 0.9472 0.1239 Slc35d2 Solute carrier family 35, member D2 0.9777 0.0458 Slc35d3 Solute carrier family 35, member D3 NA NA Slc38a1 Solute carrier family 38, member 1 0.8412 0.1757 Slc38a2 Solute carrier family 38, member 2 0.9630 0.0507 Slc38a3 Solute carrier family 38, member 3 NA NA Slc38a5 Solute carrier family 38, member 5 NA NA Slc38a7 Solute carrier family 38, member 7 0.7895 -0.2652 Slc6a14 Solute carrier family 6, member 14 NA NA Slc6a15 Solute carrier family 6 (neurotransmitter transporter), member 15 NA NA Slc6a19 Solute carrier family 6 (neurotransmitter transporter), member 19 0.9964 -0.0423 Slc7a5 Solute carrier family 7 member 5 0.3858 -0.4038 Slc7a6 Solute carrier family 7 member 6 0.9995 -0.0005 Slc7a8 Solute carrier family 7 member 8 NA 0.5053 Taldo1 Transaldolase 1 0.7591 -0.2029 Tkt Transketolase 0.7546 -0.2091 Uap1 UDP-N-acetylglucosamine pyrophosphorylase 1 0.7297 0.2763 Uap1l1 UDP-N-acteylglucosamine pyrophosphorylase 1-like 1 0.9271 -0.1403

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Chapter 3

Conclusions and Future Directions

100

Aging is inevitable, resulting in a time-dependent decline in immune function. With advances in modern technology in healthcare, life expectancy is increasing and populations in the most developed countries of the world are rapidly aging. In recent years however, there have been growing empirical evidence, based on animal models, suggesting that the aging process could be delayed and immune function can be rejuvenated among the elderly1. Indeed, we have demonstrated that age-associated increase of N-glycan branching, via increased supply of GlcNAc and IL-7 signaling, contributes to impaired T cell immunity in females. Reversing this phenotype rejuvenates

T cell responses, providing a novel therapeutic target for immune senescence and diseases that target the elderly.

An important question is what leads to the age associated increases in serum

GlcNAc. Serum GlcNAc is excreted in the kidney and therefore this may track with kidney

+ function. However, this will require formal testing. Serum GlcNAc and/or CD4 TN cell N- glycan branching level may serve as useful biomarkers of T cell aging. In future work it will be important to determine whether variability in serum GlcNAc and/or N-glycan branching correlates with variability in clinical immune function or vaccine responses in the elderly.

Aging and N-glycosylation in cancer immunotherapy

Along with an increase in life expectancy is a shift of an increasing proportion of individuals with cancer2. Among cancer immunotherapies, neutralizing antibodies that target immune checkpoints CTLA-4 and PD-1 are particularly successful3,4. Although these antibodies have resulted in dramatic improvements in disease outcome, a majority

101 of advanced stage cancer patients do not response or will relapse. Although cancer is known as a disease of old age where most cancer diagnoses are from elderly patients above the age of 655-7, a majority of studies have shown that age limits activity of anti-

PD1 and anti-CTLA-4 immunotherapy, diminishing their efficiency and effect8,9. N-glycan branching promotes PD-1 and CTLA-4 surface expression on blasting T cells10. Age- dependent increase in N-glycan branching would suggest an antagonistic effect against immune check point inhibitors such as anti-PD1 and anti-CTLA-4. Supplementing these immune check point blocking treatments with agents that target N-glycan branching may improve efficiency. We have previously shown that age-induced increases in N-glycan branching functionally suppresses T cell activity. Reversing age-dependent increase in

N-glycan branching will restore T cell function by promoting TCR clustering and signaling, reduce surface expression of PD-1 and CTLA-4, and promote pro-inflammatory TH1 and

TH17 differentiation over anti-inflammatory TH2 and Treg. Together with our findings of age-dependent increase in N-glycan branching merits exploring the effect of these agents in coordination with immune check point therapy.

Aging, IL-7, and N-glycosylation: how it impacts COVID-19 symptoms

Introductory studies produced in identifying conditions common among patients with the most severe COVID-19 symptoms narrowed down to those who are older (above

65 years in age)11, male12, or with lung conditions including asthma13,14. An early analysis that investigated a little more than 1500 patients concluded that patients with asthma were no more likely than those without asthma to be hospitalized14. However, the study didn’t investigate whether different types of asthma have different levels of risk. Thus another

102 recent study analyzed two asthma subtypes – allergic and non-allergic asthma – as separate risk factors13. The population-based study which analyzed medical records from

492,768 individuals found that non-allergic asthma significantly heightened the likelihood of severe COVID-19 whereas allergic asthma had no statistically significance associated with severe COVID-1913.

IL-2 and IL-7, which are responsible for expansion and differentiation of various T cell subsets, are increased in the sera of patients with COVID-1915, possibly due to lymphopenia increasing cytokine availability16-18. Recent studies have identified TSLP as a novel molecular player in non-allergen induced inflammatory conditions19,20. Our studies suggest that, when comparing to elderly women, men have less IL-7 signaling in T cells which results in a lack of negative feedback of IL-7Rα surface expression which may increase TSLP signaling to both suppress the development of tolerance to allergens

21-23 within the lungs while enhancing pro-inflammatory TH2 responses .

Currently research is being focused primarily on the coronavirus transmembrane spike (S) glycoprotein, which extends from the viral surface to mediate host cell entry.

This results in a crosstalk between the virus S-glycoprotein to the ACE2 receptor on the surface of human cells. With 66 N-linked glycosylation sites on the S-glycoprotein, the glycan diversity allows the pathogen to escape immune system response24,25.

Controversial antimalarial drug chloroquine, and its alternative hydroxychloroquine, have shown effectiveness in vitro in inhibiting SARS-CoV-2 infection by altering glycosylation profiles of the ACE2 receptor, thus interfering with host-cell binding and entry and its subsequent viral replication, although no effect is seen among actual COVID-19 patients26,27. Perhaps higher levels of N-glycosylation on naïve immune cells in elderly

103 women may be responsible in interfering with the viral S-glycoprotein binding to the host

ACE2 receptor, thus resulting in less severe infection and symptoms when compared to men. Further studies are required to understand the role of glycosylation on these viral and host proteins if we are to successfully diagnose, treat, and eventually prevent COVID-

19.

Summary

It is now prevalent that N-glycan branching is dynamically regulated with age and plays a role in immune senescence, however further studies are required to understand the extent of N-glycosylation and its role in regulating viral and vaccine response, cancer immunotherapy, and general immune response. Indeed, there are many age-related diseases and disorders in which we do not have a good understanding of what causes it or how it progresses. With the discovery that N-glycan branching increases with age secondary to up-regulation of IL-7 availability to naïve CD4+ T cells in females, it is increasingly important to explore glycosylation as a factor in age-related diseases and disorders. Furthermore, these treatments must take into consideration differences in sex in order to appropriately customize treatment to individuals.

104

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